Native Polyacrylamide Gel Electrophoresis (PAGE)

What is Native Polyacrylamide Gel Electrophoresis?

  • Native Polyacrylamide Gel Electrophoresis (Native Page) is a protein separation and analysis process based on charge and size. Unlike other gel electrophoresis procedures, such as SDS-PAGE, Native Page does not require denaturing chemicals in the gel matrix, such as SDS (sodium dodecyl sulfate). It instead relies on proteins’ intrinsic shape and inherent charge properties.
  • The charge of a protein in Native Page is defined by the side chains of its constituent amino acids. If the side chains are negatively charged, the protein will be negatively charged overall, and vice versa. Proteins keep their three-dimensional shape thanks to a variety of linkages, including disulfide bonds, hydrophobic interactions, and hydrogen bonds. Proteins are sorted depending on their molecular structure and charge distribution when Native Page is conducted at a neutral pH. As a result, Native Page is an extremely sensitive approach for detecting changes in protein charge or shape.
  • Because the native conformation is not altered during the process, one of the key advantages of Native Page is that the proteins studied may be retrieved in their natural state following the gel analysis. This is very valuable when examining protein-protein interactions or biological activity preservation. Furthermore, Native Page is a reasonably high-throughput approach that provides enhanced protein stability.
  • Following the completion of the gel run, the Native Page gel can be seen by staining with dyes such as bromophenol blue or other suitable staining reagents. This permits the proteins in the sample to be identified and characterized. Native Page is used in a variety of applications, such as the separation of acidic proteins, the study of glycoproteins such as human recombinant erythropoietin, and the identification of proteins such as those found in Bovine Serum Albumin (BSA).
  • An optimal approach can be used to conduct Native Page employing particular gradient gels, such as PhastGel gradient media and PhastGel native buffer strips. Gradient gels aid in the sharpening of protein bands and the separation of complicated protein mixtures on a single gel. The PhastSystem, a gel electrophoresis technology, provides quick, reproducible, and convenient Native Page separations in around 60 minutes. The system enables fine management of the running conditions while eliminating the requirement for buffer preparation.
  • While Native Page is an effective technique for analyzing the composition and structure of native proteins, it is not often utilized for measuring molecular weight. SDS-PAGE is typically thought to be more reliable and simpler for this purpose. This is because it can be difficult to locate standard proteins that closely mirror the structure, partial specific volume, and hydration properties of the natural protein under investigation.
  • Finally, Native Polyacrylamide Gel Electrophoresis (Native Page) is a useful technique for assessing proteins while maintaining their native structure and biological activity. It separates proteins based on their charge and size, and its benefits include the recovery of proteins in their original state, high throughput capabilities, and increased protein stability. Native Page can deliver fast and reproducible results by using refined techniques and gradient gels, making it a powerful technique in protein analysis and characterisation.

Principle of Native Polyacrylamide Gel Electrophoresis (PAGE)

The principle of Native Polyacrylamide Gel Electrophoresis (PAGE) is based on the separation of proteins according to their net charge, size, and shape in their native structure. The technique takes advantage of the fact that most proteins carry a net negative charge in alkaline running buffers. The migration of proteins during electrophoresis is influenced by both their charge density and the sieving effect of the gel matrix.

Proteins with higher negative charge densities, meaning more charges per unit mass, tend to migrate faster in the electric field. This is because they experience a greater electrostatic repulsion and are propelled towards the positive electrode. At the same time, the gel matrix exerts a frictional force that regulates the movement of proteins based on their size and three-dimensional shape. Smaller proteins encounter less frictional force and thus migrate more quickly, while larger proteins experience greater frictional resistance and migrate more slowly.

Therefore, native PAGE separates proteins based on a combination of their charge, mass, and structural characteristics. Since no denaturants are used, the subunit interactions within multimeric proteins are generally retained, providing insights into their quaternary structure. Additionally, some proteins maintain their enzymatic activity following separation by native PAGE, allowing for the preparation of purified, active proteins.

To perform native PAGE, the same discontinuous chloride and glycine ion fronts used in SDS-PAGE are employed to create moving boundaries that stack and separate polypeptides based on their charge-to-mass ratio. Proteins are prepared in a non-reducing and non-denaturing sample buffer, which preserves their secondary structure and native charge density. It’s important to note that in native PAGE, most proteins have an acidic or slightly basic isoelectric point (pI) ranging from approximately 3 to 8. As a result, they migrate towards the negative pole during electrophoresis. However, if a protein’s pI is higher than 8 or 9, the anode and cathode positions may need to be reversed for optimal separation.

In summary, the principle of native PAGE relies on the separation of proteins based on their native charge, size, and shape. By preserving the native structure and charge characteristics of proteins, valuable information about their subunit interactions, quaternary structure, and enzymatic activity can be obtained. Native PAGE is a useful technique for the preparation and analysis of active proteins.

Types of Native Polyacrylamide Gel Electrophoresis

There are several types of Native Polyacrylamide Gel Electrophoresis (PAGE) techniques that are commonly used for protein characterization and analysis. Here are three types of Native PAGE:

  1. Blue Native PAGE (BN-PAGE): BN-PAGE is a widely used method for the isolation and analysis of protein complexes. It allows for the one-step isolation of protein complexes from biological membranes, total cell and tissue homogenates. BN-PAGE is valuable for determining native protein masses, oligomeric states, and identifying protein-protein interactions. The native complexes recovered from gels can be further analyzed using techniques such as 2D crystallization, electron microscopy, in-gel activity assays, native electroblotting, and immunodetection.
  2. Clear Native PAGE (CN-PAGE): CN-PAGE is used to separate acidic water-soluble and membrane proteins with pI values below 7. It employs an acrylamide gradient gel and typically has lower resolution compared to BN-PAGE. The migration distance of proteins in CN-PAGE depends on their intrinsic charge and the pore size of the gradient gel. This can make estimation of native masses and oligomerization states more challenging compared to BN-PAGE. However, CN-PAGE offers advantages in cases where Coomassie dye used in BN-PAGE interferes with downstream analysis techniques, such as determination of catalytic activities or microscale separation of membrane protein complexes for fluorescence resonance energy transfer (FRET) analyses. CN-PAGE is milder and can retain labile supramolecular assemblies of membrane protein complexes that are dissociated under the conditions of BN-PAGE.
  3. Quantitative Preparative Native Continuous PAGE (QPNC-PAGE): QPNC-PAGE is a specialized form of Native PAGE that is used for the separation of metal proteins. The gel in QPNC-PAGE is polymerized for an extended period to create a gel with large pores, minimizing the molecular sieving action during electrophoretic separation. This technique allows for the isolation of metal proteins without decomposition of the metal cofactor from the apo protein. QPNC-PAGE is particularly useful for studying the structure-function relationship of metal proteins and their role in biological processes. It is applied to separate acidic, alkaline, and neutral metal proteins within a specific molecular weight range and has applications in the analysis of clinical samples where incorrect metal binding may lead to protein misfolding.

Each type of Native PAGE technique offers unique advantages and is suitable for specific applications. Researchers should consider the specific requirements of their study and choose the appropriate Native PAGE technique accordingly.

1. Blue native polyacrylamide gel electrophoresis – BN-PAGE

  • Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a popular approach for characterizing proteins in their enzymatically active state because it provides high-resolution separation. Using Coomassie Blue-G250 dye, protein complexes are observed and given an external negative charge in BN-PAGE.
  • Unlike traditional SDS-PAGE, which separates proteins by charge-to-mass ratio, BN-PAGE separates complexes by molecular weight. The complexes move through the gradient gel based on pore size until they reach the pore size limit point.
  • Coomassie Blue-G250 dye has a number of advantages over SDS. Because of its anionic nature, it can coat complexes with a negative charge without denaturing. The dye is extremely soluble in water and has an unusual capacity to attach to membrane proteins, making it ideal for membrane protein separation. The dye’s charge distribution reduces the likelihood of membrane protein aggregation. Furthermore, the interaction between the dye and membrane proteins promotes hydrophobicity loss, eventually making them water-soluble.
  • The concentration range of acrylamide and the quality of the gradient gel determine the resolution of BN-PAGE, which ranges from 100 kDa to 10 MDa. To get the best results, utilize low quantities of mild detergents like digitonin or dodecylmaltoside during the solubilization process, and consider the type of sample used for protein extraction.
  • Native proteins and complexes migrate as blue spots or bands through the gradient gel during the BN-PAGE separation procedure. After solubilization and centrifugation of the material, the addition of Coomassie Blue-G250 to the lysate aids in the visualization and characterization of the protein complexes.
  • To summarize, BN-PAGE is a strong approach for high-resolution protein separation and characterisation in their enzymatically active state. The Coomassie Blue-G250 dye is essential for viewing and producing a negative charge on protein complexes. BN-PAGE has advantages over SDS-PAGE, including the capacity to separate complexes based on molecular weight and the ability to fractionate membrane proteins. The resolution of BN-PAGE is affected by parameters such as acrylamide concentration range and gradient gel quality, while the use of mild detergents and adequate sample selection improves the findings produced.

Advantages of BN-PAGE

BN-PAGE (Blue Native Polyacrylamide Gel Electrophoresis) offers several advantages that make it a valuable technique in protein analysis:

  1. Non-denaturing Conditions: BN-PAGE allows the study of proteins under non-denaturing conditions. This means that the proteins retain their native conformation and enzymatic activity, making it possible to analyze proteins in their functionally active state. Transient interactions between proteins can also be investigated using BN-PAGE, providing insights into dynamic protein complexes.
  2. Subunit Composition Analysis: Two-dimensional BN-PAGE is a powerful tool for studying the subunit composition of protein complexes. By combining BN-PAGE with another technique, such as SDS-PAGE or immunoblotting, it is possible to gain information about the individual subunits that make up a protein complex. This information can be crucial for understanding the structure and function of the complex.
  3. Comparative Protein Associations: BN-PAGE is particularly useful for studying changes in protein associations under different experimental conditions. By comparing the protein complexes formed under different treatments or in different samples, researchers can gain insights into how protein-protein interactions are affected. This allows for the investigation of protein complex dynamics and the identification of proteins that interact under specific conditions.
  4. Confirmation of Immunoprecipitation Results: Immunoprecipitation is a commonly used technique to isolate specific protein complexes. BN-PAGE can be used as a complementary method to confirm the results of immunoprecipitation. By subjecting the immunoprecipitated complex to BN-PAGE, researchers can visualize and verify the presence of the desired protein complex, providing additional evidence for the interaction.

In summary, the advantages of BN-PAGE include the preservation of protein native conformation and enzymatic activity, the ability to analyze transient protein interactions, the identification of subunit composition in protein complexes, the study of comparative protein associations under different conditions, and the confirmation of immunoprecipitation results. These advantages make BN-PAGE a valuable technique in protein characterization and provide valuable insights into protein complex assembly and function.

Limitations of BN-PAGE

Despite its advantages, BN-PAGE (Blue Native Polyacrylamide Gel Electrophoresis) has certain limitations that researchers should be aware of:

  1. Antibody Compatibility: One limitation of BN-PAGE is that not all antibodies that work effectively in SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) may provide satisfactory results in BN-PAGE. It requires good quality and robust antibodies specifically designed for detection in native conditions. Therefore, careful selection and optimization of antibodies are necessary to ensure successful protein detection and analysis.
  2. Resolution of Protein Complexes: The resolution between different protein complexes in BN-PAGE can be relatively low. Achieving optimal resolution requires careful optimization of the gradient gel conditions, including the acrylamide concentration and gradient range. It may be necessary to adjust these parameters for each specific protein complex to obtain the desired separation and resolution.
  3. Coomassie Dye Interaction: Coomassie Blue-G250 dye, commonly used in BN-PAGE for visualization and induction of negative charge, can interact with certain protein-protein complexes. This interaction can result in smearing or obscuring of the bands on the gel, making it difficult to accurately resolve and analyze specific protein complexes. In such cases, an alternative method called CN-PAGE (Colorless Native Polyacrylamide Gel Electrophoresis) may be preferred, as it eliminates the potential interference caused by Coomassie dye.

It is important to consider these limitations when planning and interpreting experiments involving BN-PAGE. Careful optimization of experimental conditions, including antibody selection, gel gradient optimization, and consideration of alternative techniques, can help overcome some of these limitations and enhance the reliability and accuracy of BN-PAGE analysis.

2. Clear-native PAGE – CN-PAGE

  • CN-PAGE (Clear-Native Polyacrylamide Gel Electrophoresis) is a technique used for the separation of acidic water-soluble and membrane proteins with a pI (isoelectric point) below 7. While CN-PAGE typically has lower resolution compared to BN-PAGE (Blue Native Polyacrylamide Gel Electrophoresis), it offers distinct advantages in certain situations.
  • In CN-PAGE, proteins migrate through an acrylamide gradient gel, and their migration distance is determined by their intrinsic charge and the pore size of the gel. Unlike BN-PAGE, which utilizes negatively charged Coomassie dye to induce a charge shift on the proteins, CN-PAGE relies solely on the intrinsic charge of the proteins. This can make it more challenging to estimate native masses and oligomerization states compared to BN-PAGE.
  • Despite its lower resolution, CN-PAGE is preferred in specific cases where Coomassie dye interferes with subsequent analysis techniques or when specific advantages are desired. For example, CN-PAGE is useful when determining the catalytic activities of proteins or when performing efficient microscale separation of membrane protein complexes for fluorescence resonance energy transfer (FRET) analyses.
  • CN-PAGE is considered a milder technique compared to BN-PAGE. In particular, when combined with digitonin, CN-PAGE can retain labile supramolecular assemblies of membrane protein complexes that would dissociate under the conditions of BN-PAGE.
  • An important application of CN-PAGE is the identification of enzymatically active oligomeric states of mitochondrial ATP synthase that were not detected using BN-PAGE. This highlights the utility of CN-PAGE in uncovering previously unseen protein conformations and activities.
  • While BN-PAGE is commonly used for standard analyses due to its higher resolution, CN-PAGE offers specific advantages in cases where Coomassie dye interference or the preservation of labile protein complexes are of concern. By leveraging its milder conditions, CN-PAGE expands the repertoire of techniques available for studying native protein complexes.

Advantages of CN-PAGE

  1. Milder Conditions: CN-PAGE utilizes milder conditions compared to BN-PAGE, making it suitable for the preservation of labile protein complexes. This is particularly beneficial when studying delicate protein-protein interactions or enzymatically active states that may be disrupted under harsher conditions.
  2. Compatibility with Downstream Analysis: CN-PAGE can be advantageous when subsequent analysis techniques are hindered by the presence of Coomassie dye used in BN-PAGE. For example, if the detection method or assay relies on specific optical properties or requires the absence of interfering substances, CN-PAGE can be a better choice.
  3. Microscale Separation: CN-PAGE allows for efficient microscale separation of membrane protein complexes, which is useful for downstream analyses such as fluorescence resonance energy transfer (FRET). It enables the study of protein interactions and structural dynamics in a more controlled and precise manner.

Limitations of CN-PAGE

  1. Lower Resolution: CN-PAGE typically has lower resolution compared to BN-PAGE. This can make it more challenging to estimate native masses and oligomerization states of proteins accurately. If high-resolution separation is crucial for the study or identification of protein complexes, BN-PAGE may be a preferred option.
  2. Limited Applicability: CN-PAGE is primarily suitable for acidic water-soluble and membrane proteins with pI values below 7. It may not be the most suitable technique for proteins with higher pI values or certain types of complexes. Researchers should consider the specific characteristics of their target proteins before choosing CN-PAGE.
  3. Antibody Dependence: Like other gel electrophoresis techniques, CN-PAGE relies on antibodies for protein detection. The success of CN-PAGE relies on the availability of good quality and robust antibodies that can provide satisfactory results. Not all antibodies that work well in SDS-PAGE may perform optimally in CN-PAGE.
  4. Limited Comparative Analyses: CN-PAGE may not be ideal for comparative protein association studies under different experimental conditions. The lack of charge shift induced by Coomassie dye in CN-PAGE makes it more challenging to compare and analyze protein complexes directly. BN-PAGE is commonly preferred for standard comparative analyses.

It is essential to consider these advantages and limitations when deciding whether to employ CN-PAGE for protein characterization and analysis, ensuring it aligns with the specific requirements and goals of the study.

Procedure of Native Polyacrylamide Gel Electrophoresis


  1. Coomassie Brilliant Blue G-250 Solution:
    • Dissolve 2.5 mg of Coomassie Brilliant Blue in 0.45 mL of methanol.
    • Add 0.45 mL of deionized water.
    • Add 0.1 mL of glacial acetic acid.
  2. Acrylamide Solution (40%):
    • Dissolve 20 g of acrylamide and 5.3 g of bis-acrylamide in deionized water to a final volume of 50 mL.
  3. Separating Gel Buffer (4×):
    • Dissolve 18.15 g of Tris in about 75 mL of deionized water.
    • Adjust the pH to 8.8 with HCl.
    • Add deionized water to a final volume of 100 mL.
  4. Ammonium Persulfate Solution (10%):
    • Dissolve 0.1 g of ammonium persulfate in a final volume of 1 mL of deionized water.
  5. Sample Solution (2×):
    • Prepare a solution containing 0.187 M Tris/HCl (pH 6.8), 30% glycerol, and 80 μg/mL of Bromophenol Blue.
  6. Electrophoresis Buffer:
    • Dissolve 28.8 g of glycine and 6 g of Tris in deionized water to a final volume of 2 L.

Preparation of a Continuous (10%) Polyacrylamide Gel for Native and Blue Native Gel Electrophoresis

Here is a description of the preparation steps for a continuous (10%) polyacrylamide gel for Native and Blue Native gel electrophoresis:

  1. Start by thoroughly cleaning and drying the glass plates, suitable spacers, and comb. Assemble the glass plates, spacers, and comb according to the manufacturer’s instructions.
  2. In a suitable container, mix 2.5 mL of acrylamide solution (40%), 2.5 mL of separating gel buffer (4×), and 5 mL of deionized water. Ensure proper mixing of the components. Note that the concentrations mentioned here are based on the specific requirements for a 10% polyacrylamide gel.
  3. Degas the mixture to eliminate air bubbles from the gel after polymerization and remove oxygen, which can speed up the polymerization process. This can be achieved by applying a vacuum or using a suitable degassing apparatus.
  4. Add 50 μL of ammonium persulfate solution (10%) and 10 μL of TEMED to the gel mixture. These compounds initiate the polymerization process. Again, ensure thorough mixing to distribute these components evenly.
  5. Pour the mixed solution between the glass plates, taking care not to introduce air bubbles. Place the comb in the appropriate position to create wells for sample loading.
  6. Allow the acrylamide mixture to polymerize for approximately 1 hour. During this time, the gel solidifies, forming a continuous polyacrylamide matrix.
  7. Carefully remove the comb from the gel, ensuring that the wells are clear and ready for sample loading. The gel is now prepared and can be used for Native or Blue Native gel electrophoresis experiments.

It’s important to note that the specific volumes and concentrations mentioned in this description may vary depending on the desired gel percentage and experimental requirements. Always refer to the manufacturer’s instructions or established protocols for precise guidelines.

Sample Preparation

Here are the steps for sample preparation for Native PAGE and Blue Native PAGE:

Sample Preparation for Native PAGE:

  1. Take 10 μL of the protein solution that you want to analyze.
  2. Mix the protein solution with 10 μL of sample solution (2×). The sample solution should contain glycerol and the dye Bromophenol Blue.
  3. Thoroughly mix the protein and sample solution to ensure uniform distribution of the components.
  4. The sample is now ready for loading onto the Native PAGE gel for electrophoresis. Proceed with the gel loading according to the specific gel running protocol.

Sample Preparation for Blue Native PAGE:

  1. Take 10 μL of the protein solution that you want to analyze.
  2. Mix the protein solution with 10 μL of sample solution (2×). The sample solution should contain glycerol, the dye Bromophenol Blue, and also Coomassie Brilliant Blue solution.
  3. Thoroughly mix the protein, sample solution, and Coomassie Brilliant Blue solution to ensure proper staining and visualization of the protein complexes.
  4. The sample is now ready for loading onto the Blue Native PAGE gel for electrophoresis. Follow the gel running protocol specific to Blue Native PAGE for optimal separation and analysis of protein complexes.

It’s important to note that the specific volumes and concentrations mentioned in this description may vary depending on the experimental requirements and protocols being followed. Adjustments may be necessary based on the protein concentration and the specific sample buffer used in your experiment.

Marker Sample

To prepare a marker sample for Native PAGE, you can follow these steps:

  1. Obtain a prestained protein marker that is commonly used for SDS-PAGE. These markers contain a mixture of proteins with known molecular weights.
  2. Thoroughly mix the marker solution by gently inverting the tube to ensure uniform distribution of the proteins.
  3. Determine the appropriate volume of the marker solution to load onto the gel. This can vary depending on the size of the wells and the desired intensity of the marker bands.
  4. Pipette the desired volume of the marker solution into a separate microcentrifuge tube.
  5. If the marker solution contains glycerol or any other additives that are incompatible with the Native PAGE running buffer, dilute the marker solution with an appropriate buffer compatible with Native PAGE (such as sample buffer or running buffer).
  6. Mix the diluted marker solution by gently inverting the tube to ensure proper mixing of the components.
  7. The marker sample is now ready for loading onto the gel. Load the marker sample into a dedicated well alongside your sample wells.
  8. Proceed with the gel running protocol for Native PAGE, applying the appropriate voltage and running time.

By including a marker sample in your Native PAGE experiment, you will be able to estimate the approximate molecular weights of the protein bands in your sample by comparing them to the migration pattern of the marker proteins.

(Blue) Native PAGE Conditions

One dimensional eletrophoresis of BSA.
One dimensional eletrophoresis of BSA.

The (Blue) Native PAGE conditions involve the following steps:

  1. Fill the electrophoresis apparatus with the appropriate gel electrophoresis buffer. The buffer should be compatible with Native PAGE and provide optimal conditions for protein migration.
  2. Start the electrophoresis immediately after assembling the gel in the apparatus. The recommended voltage and current settings may vary depending on the gel thickness and length. For a gel with 1 mm thickness and 15 cm length, applying around 150 V (constant voltage) should result in approximately 20 mA of current. It is important to refer to the specific instructions or protocols for your experimental setup (see Notes 4 and 5).
  3. Once the electrophoresis is complete, carefully remove the gel from between the glass plates.
  4. Following native gel electrophoresis, the gel can be stained to visualize the protein bands. Two commonly used staining methods are Coomassie Brilliant Blue (Fig. 1a) and silver staining (Fig. 1b). Choose the staining method that best suits your experimental needs and follow the corresponding staining protocol.
  5. After staining, the blue native gel should be destained to remove any excess dye and enhance the visibility of the protein bands. Destaining is typically done using deionized water (Fig. 1c) (see Note 6).

By following these (Blue) Native PAGE conditions, you can separate and visualize protein complexes or native protein assemblies based on their size and charge. The stained and destained gel will provide valuable information about the composition and organization of the proteins under investigation.


  1. Water Quality: Unless specified otherwise, use water with a resistance greater than 18 MΩ and a total organic content of less than five parts per billion. This high-quality water, referred to as “water” in this context, ensures the purity of the solutions.
  2. Fresh Preparation: Prepare the necessary solutions, such as staining or gel solutions, immediately before use. Freshly prepared solutions yield more reliable results.
  3. Adjusting Volume: Adjust the volume of the gel solution according to the requirements of your specific gel equipment. Ensure you have the appropriate amount of solution to achieve the desired gel thickness and size.
  4. Current Fluctuation: During electrophoresis with constant voltage, the current will gradually decrease over time. This phenomenon is normal and should be taken into account when monitoring the progress of the electrophoresis process.
  5. Bromophenol Blue Dye Front: The migration of the Bromophenol Blue dye front, which indicates the progress of electrophoresis, typically takes around 3 hours to reach the bottom of the gel. Higher voltages can accelerate electrophoresis but may generate more heat within the gel. Find the balance between speed and heat generation based on your experimental needs.
  6. Native Bovine Serum Albumin (BSA): BSA is a protein with a complex, heart-like structure stabilized by disulfide bridges. Native PAGE can partially unfold BSA and separate its different forms based on their mobility. This property of BSA makes it a useful protein for studying native PAGE techniques (references 3 and 4).

Considering these notes will help ensure the accuracy, reliability, and appropriate conditions for your experiments involving native and blue native PAGE.

Buffers for Native-PAGE

Buffers play a crucial role in Native-PAGE (polyacrylamide gel electrophoresis) for the separation of proteins based on their native states. Here are the recommended buffers for Native-PAGE:

  1. Stacking Gel Buffer (for a 5 ml gel):
  • 0.375 M Tris-HCl, pH 8.8: 4.275 ml
  • Acrylamide/Bis-acrylamide (30%/0.8% w/v): 0.67 ml
  • *10% (w/v) ammonium persulfate (AP): 0.05 ml
  • TEMED: 5 μl ( Added right before each use)
  1. Separating Gel Buffer (for a 10 ml gel):
  • Acrylamide percentage:
    • 6%: Acrylamide/Bis-acrylamide (30%/0.8% w/v): 2 ml
    • 8%: Acrylamide/Bis-acrylamide (30%/0.8% w/v): 2.6 ml
    • 10%: Acrylamide/Bis-acrylamide (30%/0.8% w/v): 3.4 ml
    • 12%: Acrylamide/Bis-acrylamide (30%/0.8% w/v): 4 ml
    • 15%: Acrylamide/Bis-acrylamide (30%/0.8% w/v): 5 ml
  • 0.375 M Tris-HCl, pH 8.8:
    • 6%: 7.89 ml
    • 8%: 7.29 ml
    • 10%: 6.49 ml
    • 12%: 5.89 ml
    • 15%: 4.89 ml
  • *10% (w/v) ammonium persulfate (AP): 100 μl
  • TEMED: 10 μl ( Added right before each use)
  1. Sample Buffer (2x):
  • 62.5 mM Tris-HCl, pH 6.8
  • 25% glycerol
  • 1% Bromophenol Blue
  1. Running Buffer:
  • 25 mM Tris
  • 192 mM glycine Note: The running buffer should be approximately pH 8.3. Do not adjust the pH.

It’s important to note that these buffer recipes are provided as a guideline, and slight modifications may be necessary based on specific experimental conditions or requirements.

Gel running protocol

Here is a gel running protocol for Native-PAGE, based on the provided information:

  1. Preparation of Separating Gel: a. Prepare the appropriate amount of separating gel solution in a small beaker. b. Add the specific volume of ammonium persulfate (AP) and N,N,N’,N’-Tetramethylethylenediamine (TEMED) to the gel solution. c. Gently swirl the beaker to ensure thorough mixing. d. Pipette the gel solution into the gap between the glass plates of the gel casting apparatus, taking care not to overfill it. e. Fill the remaining space in the apparatus with water (or isopropanol as an alternative). f. Allow 20-30 minutes for complete gelation.
  2. Preparation of Stacking Gel: a. While the separating gel is gelating, prepare the appropriate amount of stacking gel solution in a beaker. b. Mix the stacking gel solution with 10% ammonium persulfate (AP) and 1% TEMED. c. Pour out the water or isopropanol from the previous step. d. Pipette the stacking gel solution into the gap created in step 2f. e. Insert the comb into the stacking gel solution. f. Allow 20-30 minutes for the stacking gel to gelate.
  3. Sample Preparation: a. Mix your protein sample with sample buffer. b. Do not heat your sample, as Native-PAGE preserves the native structure of proteins.
  4. Loading and Electrophoresis: a. Carefully remove the comb from the gel apparatus. b. Load the prepared sample mixture into the wells of the gel. c. Set an appropriate voltage to run the electrophoresis. Note: It is recommended to place the gel apparatus on ice during the run and avoid setting a high voltage to prevent protein degradation.
  5. Staining or Western Blotting: a. Once the electrophoresis is complete, you can proceed with staining using a standard Coomassie Blue staining protocol. b. Alternatively, if you intend to perform immunoblotting (Western blotting), transfer the separated proteins onto a membrane for further analysis.

It’s important to note that this protocol provides a general guideline for gel running in Native-PAGE. Additional modifications or steps may be required based on specific experimental conditions or desired applications.

Native-PAGE Staining Methods

Native-PAGE staining methods offer different sensitivities and characteristics for visualizing protein bands. Here are the procedures for Coomassie Blue staining, Silver staining, and Fluorescent dye staining:

  1. Coomassie Blue (G-250) staining:
  • Sensitivity: 0.2~0.5 μg/band

Staining solution:

  • 0.3% Coomassie Brilliant Blue R-250 (w/v)
  • 45% Methanol (v/v)
  • 10% Glacial acetic acid (v/v)
  • 45% Water

Destaining solution:

  • 20% Methanol (v/v)
  • 10% Glacial acetic acid (v/v)
  • 70% Water


  1. Immerse the gel in the staining solution and gently shake it on a horizontal rotator for approximately 20-30 minutes.
    • Optionally, you can microwave the gel for 1-3 minutes, but be careful not to exceed 10 seconds to prevent boiling.
  2. Transfer the gel to the destaining solution and shake it on the same apparatus for about 20-30 minutes.
    • Change the destaining solution 3-5 times until clear bands with minimal blue background are visible.
    • Microwaving can also be used during destaining, with careful monitoring of the time.
  3. Silver staining:
  • Sensitivity: 2 ng/band


  1. Prepare a fixing solution: 50% Methanol, 10% Acetic Acid, and 50 μl Formaldehyde in a total volume of 100 ml. Immerse the gel in this solution for one hour to overnight.
  2. Wash the gel with 50% ethanol for three times, with each wash lasting no less than 20 minutes.
  3. Treat the gel with hypo solution (20 mg Sodium Thiosulfate in 100 mL) for 1 minute. Save 2 ml of this solution for a later step. Over-treating with hypo solution can result in a darker gel.
  4. Wash the gel with water for three times, with each wash lasting 20 seconds.
  5. Treat the gel with Silver Nitrite solution (200 mg in 100 mL) for 30 minutes. Avoid over-treatment, as it may lead to unwanted artifacts.
  6. Wash the gel with water for three times, with each wash lasting 20 seconds.
  7. Develop the gel in a developing solution (6 g Sodium Carbonate, 2 ml of the saved hypo solution, and 50 μl Formaldehyde) until desired band intensity is achieved. The duration depends on the protein concentrations and can range from one minute to 30 minutes. Overdeveloping may result in a dark brown gel.
  8. Once developed, stop the reaction with 5% Acetic Acid. Stop developing the gel immediately upon observing the bands.
  9. Store the gel in the fixing solution and try to dry it as soon as possible. Avoid storing the gel for too long.
  10. Fluorescent dye staining:
  • Sensitivity: 5 ng/band
  • No background, but capturing the results may be more challenging.

These staining procedures provide options for visualizing protein bands in Native-PAGE gels, with different sensitivities and characteristics. Choose the staining method that best suits your requirements.

Native PAGE Tips

When performing Native PAGE, it’s important to pay attention to certain details and follow some tips for optimal results. Here are some tips to consider:

  1. Different buffers for different pI values: Use a high-pH gel system (pH 8.0-9.0) for separating acidic proteins. Adjusting the buffer to a higher pH ensures that acidic proteins become negatively charged and migrate towards the positive anode in the gel. For separating alkaline proteins, use a low-pH gel system. Electrophoresis is typically performed in a slightly acidic buffer environment, so inverting the cathode and anode is necessary to separate alkaline proteins effectively.
  2. Loading considerations: Ensure that the ionic intensity of your sample is not higher than 0.1 mmol/L to prevent deformation of the electrophoretic bands. Centrifuge your sample before loading to prevent loading any pellet that might interfere with the analysis. In most cases, a marker is not necessary for native PAGE. However, if you require a marker, make sure to use specific native PAGE markers designed for this purpose.
  3. Gel running: Perform a pre-run for 30-60 minutes before loading your samples. This allows for equilibration of the gel and removal of any impurities. Pay attention to the voltage used for gel running. Generally, the voltage should not be lower than 100V or higher than 200V. Adjust the voltage accordingly to ensure efficient migration of the proteins.

By following these tips, you can optimize your Native PAGE experiments and obtain reliable and accurate results.


SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and Native PAGE (Native Polyacrylamide Gel Electrophoresis) are two different electrophoretic techniques used to separate proteins based on different principles. Here are some key differences between SDS PAGE and Native PAGE:

  1. Description:
    • SDS PAGE: It is a technique that separates proteins primarily based on their mass. The proteins are denatured and coated with SDS (Sodium Dodecyl Sulfate), which imparts a negative charge to the proteins, causing them to migrate through the gel based on their size.
    • Native PAGE: It is an electrophoretic technique that separates proteins based on their size and charge. The proteins are not denatured during the process, preserving their native conformation and charge distribution.
  2. Nature of Gel:
    • SDS PAGE: The gel used in SDS PAGE is denatured, as the proteins are unfolded and coated with SDS.
    • Native PAGE: The gel used in Native PAGE is not denatured, maintaining the native structure and conformation of the proteins.
  3. Denaturation:
    • SDS PAGE: In SDS PAGE, SDS is added to the gel and protein samples to denature the proteins and give them a uniform negative charge.
    • Native PAGE: No denaturation step is required in Native PAGE as the proteins retain their native structure and charge distribution.
  4. Basis of Separation:
    • SDS PAGE: Proteins in SDS PAGE are separated primarily based on their mass. The SDS coating and uniform charge allow for size-based separation.
    • Native PAGE: Proteins in Native PAGE are separated based on their size and charge. The separation is influenced by both the size of the protein and its charge distribution in the native state.
  5. Protein Stability and Recovery:
    • SDS PAGE: Proteins in SDS PAGE are not stable due to denaturation, and therefore cannot be recovered in their native form after electrophoresis.
    • Native PAGE: Proteins in Native PAGE retain their stability and native conformation, allowing for subsequent recovery and analysis of the proteins.

Both SDS PAGE and Native PAGE have their specific uses and advantages. SDS PAGE is commonly used for protein size determination and quantification, while Native PAGE is suitable for studying native protein-protein interactions, protein complexes, and assessing protein conformational changes. The choice between the two techniques depends on the specific research goals and the nature of the proteins being studied.

Gel NatureDenatured gelNon-denatured gel
DenaturationSDS is used to denature proteinsNo denaturation step required
Separation PrincipleBased on protein massBased on protein size and charge
Protein StabilityProteins are not stable and cannot be recoveredProteins are stable and can be recovered
Protein ConformationProteins are unfoldedProteins retain their native conformation
Protein ActivityProtein activity is lostProtein activity is preserved
Sample PreparationRequires boiling in SDS sample bufferMinimal or no sample preparation required
ResolutionHigher resolution due to denaturationLower resolution due to native conformation
ApplicationsAnalysis of protein molecular weight and purityAnalysis of protein complexes and native structures

Applications of Native Polyacrylamide Gel Electrophoresis (PAGE)

Native Polyacrylamide Gel Electrophoresis (PAGE) is a powerful technique used in various applications to study the structure and characteristics of proteins and protein complexes. Here are some common applications of Native PAGE:

  1. Protein Complex Analysis: Native PAGE allows the separation and analysis of protein complexes based on their native size, shape, and charge. It is often used to study protein-protein interactions, oligomerization, and multimeric assembly of protein complexes.
  2. Enzyme Activity Assays: Native PAGE can be used to determine the activity of enzymes by incorporating specific substrates or cofactors into the gel. After electrophoresis, the gel is incubated under conditions suitable for enzyme activity, and the appearance of product bands indicates enzyme activity.
  3. Protein Purity and Homogeneity Analysis: Native PAGE can assess the purity and homogeneity of protein samples. It allows the detection of multiple protein species, such as different isoforms or post-translational modifications, based on their distinct migration patterns.
  4. Native Protein Localization: Native PAGE can be used to determine the subcellular localization of proteins or protein complexes. By subjecting cellular extracts to Native PAGE, the distribution of native proteins across different cellular compartments can be analyzed.
  5. Protein Conformational Studies: Native PAGE can provide insights into the conformational changes of proteins. By incorporating denaturing agents or ligands that stabilize or induce conformational changes, Native PAGE can reveal alterations in protein structure or conformational transitions.
  6. Protein-DNA/RNA Interactions: Native PAGE is useful for studying protein-DNA or protein-RNA interactions. By including nucleic acid substrates in the gel, Native PAGE can separate and analyze nucleic acid-binding proteins based on their interaction with the nucleic acid.
  7. Antibody Analysis: Native PAGE is employed in the characterization of antibodies, including the identification of antibody subclasses, determination of antibody integrity, and analysis of antibody-antigen interactions.
  8. Membrane Protein Studies: Native PAGE is particularly valuable for studying membrane proteins, which often require preservation of their native environment for proper folding and function. It allows the analysis of native membrane protein complexes and their interactions with other proteins or ligands.

Advantages of Native Polyacrylamide Gel Electrophoresis (PAGE)

  1. Analysis of Native Protein Conformation: Native PAGE allows the separation and analysis of proteins in their native conformation without denaturation. This is important for studying protein structure, function, and native protein-protein interactions.
  2. Retention of Protein Activity: Native PAGE preserves the enzymatic activity and functional properties of proteins, enabling the analysis of biologically active protein complexes and enzymes in their native state.
  3. Determination of Oligomeric States: Native PAGE can reveal the oligomeric states of proteins and protein complexes, providing insights into their quaternary structure and assembly.
  4. Study of Protein-Protein Interactions: Native PAGE allows the examination of protein-protein interactions under native conditions, providing information on protein complex formation, stability, and dynamics.
  5. Resolution of Protein Isoforms: Native PAGE can separate protein isoforms and variants based on their size, charge, or both, allowing the identification and characterization of different forms of a protein within a sample.
  6. Compatibility with Non-Denaturing Dyes and Stains: Native PAGE can be combined with non-denaturing dyes and stains, such as Coomassie Brilliant Blue or silver staining, to visualize and quantify proteins without disrupting their native structure or function.
  7. Protein Recovery for Further Analysis: Proteins separated by Native PAGE can be recovered from the gel for downstream applications, such as mass spectrometry, protein sequencing, or functional assays, while preserving their native properties.
  8. Versatility in Buffer Systems: Native PAGE can be performed using different buffer systems and pH conditions to optimize separation based on protein size, charge, or both. This allows flexibility in experimental design and customization.
  9. Compatibility with Other Protein Analysis Techniques: Native PAGE can be combined with other techniques, such as immunoblotting, immunoprecipitation, or enzyme activity assays, to further characterize and analyze proteins in their native state.
  10. Established Methodology: Native PAGE is a well-established technique with established protocols, equipment, and reagents readily available, making it accessible and reproducible for researchers.

Overall, the advantages of Native Polyacrylamide Gel Electrophoresis (PAGE) make it a valuable tool for studying native protein structure, function, interactions, and isoforms, allowing researchers to gain insights into the biology and behavior of proteins under native conditions.

Disadvantages of Native Polyacrylamide Gel Electrophoresis (PAGE)

  1. Limited Resolution: Native PAGE may have limited resolving power compared to other separation techniques, such as SDS-PAGE or high-resolution chromatography. This can result in overlapping bands or insufficient separation of closely related proteins.
  2. Difficulty in Standardizing Mobility: The mobility of proteins in native gels can be influenced by multiple factors, including size, charge, shape, and interactions with the gel matrix. This variability makes it challenging to establish standardized mobility patterns for accurate comparison between experiments or laboratories.
  3. Protein Aggregation and Precipitation: Native PAGE may induce protein aggregation or precipitation during the gel running process, particularly for proteins with high isoelectric points or hydrophobic regions. This can lead to distorted band patterns and loss of protein integrity.
  4. Lack of Denaturation and Unmasking of Epitopes: Unlike denaturing techniques, Native PAGE does not denature proteins, which can limit the accessibility of epitopes for antibody binding in immunoblotting or other immunodetection methods.
  5. Limited Detection Sensitivity: Native PAGE may have lower detection sensitivity compared to techniques like Western blotting or fluorescence-based assays. This is because native gels often lack the amplification methods used in these techniques, such as enzyme-conjugated secondary antibodies or signal amplification systems.
  6. Incomplete Disruption of Protein-Protein Interactions: Native PAGE may not fully disrupt all protein-protein interactions, particularly strong or stable complexes. This can result in the co-migration of proteins within complexes and make it difficult to distinguish individual subunits or analyze specific interactions.
  7. Time-Consuming Procedure: Native PAGE requires longer running times compared to denaturing techniques, as the separation is based on the size and charge of proteins without the aid of denaturing agents. This can increase the overall experimental time and delay result acquisition.
  8. Limited Compatibility with Protein Analysis Techniques: Certain downstream protein analysis techniques, such as mass spectrometry or protein sequencing, may require the denaturation and digestion of proteins. Native PAGE may not be compatible with these techniques, limiting the options for further characterization.
  9. Protein Loss and Diffusion: Some proteins may exhibit diffusion or loss from the gel matrix during electrophoresis, leading to distorted or incomplete protein bands. This can affect the accuracy and reliability of protein quantification and analysis.
  10. Gel-to-Gel Variability: Native PAGE can be prone to gel-to-gel variability, especially when performed manually. Factors such as gel composition, casting conditions, and buffer preparation can introduce variability between experiments or replicates.

Despite these limitations, Native PAGE remains a valuable tool for studying native protein structure, interactions, and isoforms when combined with appropriate controls, careful optimization, and complementary techniques to address its shortcomings.


What is Native Polyacrylamide Gel Electrophoresis (PAGE)?

Native PAGE is an electrophoretic technique used to separate proteins based on their size and charge under non-denaturing conditions. It preserves the native structure and function of proteins, allowing analysis of protein-protein interactions, oligomerization, and complex formation.

How does Native PAGE differ from SDS-PAGE?

Unlike SDS-PAGE, which uses denaturing agents to unfold proteins and separate them solely based on size, Native PAGE maintains the native conformation of proteins, separating them based on both size and charge. It provides information about native protein structures and interactions.

What is the purpose of Native PAGE?

Native PAGE is commonly used to study protein complexes, oligomerization, and protein-protein interactions. It helps in identifying different protein isoforms, determining native molecular weights, and investigating protein assembly/disassembly processes.

What is the gel composition used in Native PAGE?

The gel matrix used in Native PAGE typically consists of polyacrylamide and a non-ionic detergent, such as Triton X-100, to solubilize membrane proteins while preserving their native conformation.

What are the advantages of Native PAGE over other protein separation techniques?

Native PAGE allows the analysis of native protein structures and interactions without denaturation, preserving their biological activity. It can provide valuable insights into protein complexes, protein-protein interactions, and molecular weight estimation under native conditions.

How can Native PAGE be used to determine protein oligomerization?

Native PAGE separates protein complexes based on their size and charge. By comparing the migration patterns of known protein complexes with the migration of individual proteins, one can determine the oligomeric state of the protein of interest.

Can Native PAGE be used for quantification of protein samples?

Quantification of proteins using Native PAGE can be challenging due to various factors, such as variable staining or detection methods. However, relative quantification can be achieved by comparing band intensities within the same gel under standardized conditions.

Can Native PAGE be combined with other techniques for further analysis?

Yes, Native PAGE can be combined with other techniques such as immunoblotting, mass spectrometry, or activity assays to provide additional information about protein identification, post-translational modifications, or functional characterization.

What are the limitations of Native PAGE?

Some limitations of Native PAGE include limited resolution, difficulty in standardizing mobility, lower sensitivity compared to other techniques, and the potential for protein aggregation or loss during electrophoresis.

How can gel-to-gel variability in Native PAGE be minimized?

To minimize gel-to-gel variability, it is important to maintain consistent gel preparation conditions, such as gel composition, pH, buffer conditions, and handling techniques. Proper standardization and the use of appropriate controls can help in reducing variability between experiments.


  1. Arndt, C., Koristka, S., Bartsch, H., & Bachmann, M. (2012). Native Polyacrylamide Gels. Protein Electrophoresis, 49–53. doi:10.1007/978-1-61779-821-4_5 

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