Native Polyacrylamide Gel Electrophoresis (PAGE)

What is Native Polyacrylamide Gel Electrophoresis?

• Native Polyacrylamide Gel Electrophoresis (Native Page) is a protein separation and analysis technique that uses the charge and size of proteins for separation and identofication. Contrary to other gel electrophoresis techniques, such as SDS-PAGE, Native Page does not include denaturing chemicals in the gel matrix, for example, SDS (sodium dodecyl sulfate). Rather, it is conferred by the protein’s shape and charges.
• In Native Page, a protein’s charge is determined by the side chains of its amino acid residues. If the side chain is of a negative charge the protein negatively charged too, and vice-versa. Proteins are maintained in their functional conformations and shapes due to many types of linkages of primary importante are disulfide linkages, hydrophobic linkages, and hydrogen linkages. The proteins are resolved owing to their differences in the molecular shape as well as the charge distribution in the molecules when a Native Page is performed at neutral pH. Therefore, Native Page is highly sensitive method for the determination of charge shape, or other structural changes that may occur or having occurred in a protein.
• One key benefit of Native PAGE is that the proteins being analyzed can be recovered in a native form post gel examination. This is greatly beneficial in studying protein-protein interactions and biochemical activities. Additionally, Native PAGE provides improved protein stability and is fairly high throughput.
• Native PAGE gels can be visualized after running the gel by using staining techniques, which employ bromophenol blue or other compatible staining dyes. This allows for the identification and characterization of proteins within the sample. One of the many uses for Native PAGE includes the separation of proteins with an acidic isoelectric point, the analysis of human recombinant erythropoietin’s glycoproteins, and the culturing of some proteins contained in bovine serum albumin (BSA).
• Specific gradient gels, like PhastGel gradient media and PhastGel native buffer strips, are well suited for conducting Native Page with an optimal approach. These gels assist in the focusing of protein bands and the resolution of intricate mixtures of proteins within a single gel. Such a system, known as PhastSystem, allows the rapid, reproducible, and convenient completion of gel electrophoresis – a technology which facilitates Native Page separation in less than an hour. This system offers greater control over the temperature and voltage settings for sample separation while eliminating the need for buffer solutions.
• Even though measuring molecular weight is achievable through Native Page, it is not the preferred choice. For such needs, SDS-PAGE is commonly accepted, as it is simple to use and highly accurate. This is standard practice because standard proteins possessing similar structure, partial specific volume, and hydration properties to the protein being examined are generally difficult to obtain.
• Lastly, Native Page is a method of electrophoresis that is useful in analyzing the proteins while preserving their structure and biological activity. It separates proteins based on their charge and size, and its benefits incorporate protein recovery in their original form, high throughput, and enhanced stability of proteins. Using more advanced techniques and gradient gels, native page enables protein analysis and characterization through all reputable results with ease and speed. It has tremendous capabilities in delivering fast and reproducible results.

Principle of Native Polyacrylamide Gel Electrophoresis (PAGE)

An analytical technique called SDS-PAGE (Polyacrylamide Gel Electrophoresis) separates protein mixture components according to size.

The method is predicated on the idea that a charged molecule would migrate in an electric field toward an electrode of opposite sign. Since the mobility of a material in the gel depends on both charge and size, the conventional electrophoresis techniques cannot be applied to ascertain the molecular weight of biological molecules.

To circumvent this, the biological samples must be processed such that they acquire homogenous charge; then, the electrophoretic mobility depends mostly on size. Needs to be denatured (done with the use of SDS) for this various protein molecules with diverse shapes and sizes so that the proteins lose their secondary, tertiary or quaternary structure.Negative charged proteins covered by SDS migrate towards the anode (positively charged electrode) are separated by a molecular sieve action depending on size when deposited onto a gel and placed in an electric field. Comparatively with a known molecular weight ladder (marker), the size of a protein may be computed following the visualization by a staining (protein-specific) procedure by means of their migration distance.

Types of Native Polyacrylamide Gel Electrophoresis

Commonly applied for protein characterisation and analysis are numerous forms of Native Polyacrylamide Gel Electrophoresis (PAGE) methods. Three forms of Native PAGE are as follows:

• Blue Native PAGE (BN-PAGE)– BN-PAGE, or Blue Native PAGE, is a commonly used technique for protein complex separation and study. It lets one-step isolate protein complexes from biological membranes using entire cell and tissue homogenates. Determining native protein weights, oligomeric states, and spotting protein-protein interactions all benefit from BN-PAGE. Techniques include 2D crystallization, electron microscopy, in-gel activity tests, native electroblotting, and immunodetection allow one to examine the native complexes retrieved from gels further.
• Clear Native PAGE (CN-PAGE)– Using pI values less than 7, clear Native PAGE (CN-PAGE) separates acidic water-soluble and membrane proteins. Usually of poorer resolution than BN-PAGE, it uses an acrylamide gradient gel. Protein migration in CN-PAGE is determined by their inherent charge as well as gradient gel pore size. This makes estimate of native mass and oligomerization states more difficult than in BN-PAGE. When Coomassie dye used in BN-PAGE interacts with downstream analytical methods, including determination of catalytic activities or microscale separation of membrane protein complexes for fluorescence resonance energy transfer (FRET) studies, CN-PAGE provides benefits instead. Milder and able to preserve labile supramolecular structures of membrane protein complexes dissociated under BN-PAGE conditions is CN-PAGE.
• Quantitative Preparative Native Continuous PAGE (QPNC-PAGE)– Used for the separation of metal proteins, quantitative preparative native continuous PAGE (QPNC-PAGE) is a specialist variation of Native PAGE. By polymerizing the gel in QPNC-PAGE for a long duration, a gel with significant holes is produced, hence reducing the molecular sieving action during electrophoretic separation. This method lets metal proteins be isolated free from the breakdown of the metal cofactor from the apo protein. Researching the structure-function link of metal proteins and their function in biological processes benefits especially with QPNC-PAGE. It has uses in the examination of clinical samples where improper metal binding may cause protein misfolding and is employed to differentiate acidic, alkaline, and neutral metal proteins within a certain molecular weight range.

Every kind of Native PAGE method has special benefits and fits particular uses. Researchers should pick the suitable Native PAGE approach according on the particular criteria of their project.

Requirements for Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide Gel Electrophoresis (PAGE) is a widely used technique for separating proteins based on their size. To perform PAGE, several reagents, buffers, and equipment are required.

1. Reagents and Buffers

• Acrylamide solutions – Used to prepare the resolving and stacking gels.
• Isopropanol / Distilled water – Helps in gel polymerization and prevents contamination.
• Gel loading buffer – Facilitates sample loading and enhances visibility.
• Running buffer – Maintains the pH and conductivity for efficient protein migration.
• Staining and destaining solutions – Used to visualize separated protein bands after electrophoresis.
• Protein samples – The target proteins to be analyzed.
• Molecular weight markers – Reference standards for estimating protein sizes.

2. Equipment and Supplies

• Electrophoresis chamber and power supply – Essential for applying an electric field to drive protein migration.
• Glass plates (short and top plate) – Forms the gel mold and holds the polyacrylamide gel.
• Casting frame – Supports the glass plates during gel preparation.
• Casting stand – Ensures proper alignment and prevents gel leakage during casting.
• Combs – Creates wells in the gel for sample loading.

Procedure of Native Polyacrylamide Gel Electrophoresis

1. Sample Preparation
   • Protein separation is accomplished using native PAGE so that their natural structure and biological activity are preserved.
   • Unlike SDS-PAGE, no denaturing chemicals including SDS or urea are used.
   • The protein sample is made in a suitable non-denaturing buffer such that physiological pH and ionic strength are preserved.
   • Throughout electrophoresis, a tracking dye—such as bromophenol blue—may be introduced to track migration.
   • Among recent developments include the use of tailored sample buffers improving protein stability and resolution.
2. Preparation of Polyacrylamide Gel
   • Acrylamide and bisacrylamide make up the gel; polymerized in a buffer solution that maintains protein structure, they form
   • The range of molecular weight of the proteins under analysis determines the concentration of acrylamide:
   • Low percentage gels (5–7%) help to reduce highly molecular weight proteins.
   • Higher percentage gels (10–15%) fix smaller proteins.
   • Using a gel casting device, the gel is polymerized between two glass plates; a comb at the top creates sample wells.
   • The comb is taken off upon polymerization, and the gel is run-through with the running buffer.
   • Recent studies indicate that gradient gels might help to enhance the resolution and separation of complicated protein mixtures.
3. Electrophoresis
   • Usually keeping a steady pH to avoid protein denaturation, a buffer system fit for native circumstances is employed.
   • Along with a molecular weight marker, the ready protein samples are pipled into the wells.
   • Applied over the gel, an electric field causes proteins to move depending on their charge, size, and structure.
   • Unlike SDS-PAGE, in which migration is based just on molecular weight, proteins in Native PAGE move in line with their general charge and conformation.
   • Until enough separation is obtained, the gel runs for a designated duration under either continuous voltage or current.
   • Pre-cast gels and sophisticated electrophoresis chambers are now part of modern systems to improve repeatability and efficiency.
4. Detection and Analysis
   • Proteins are seen following electrophoresis by staining methods including:
     • broad protein detection using coomassie brilliant blue staining.
     • Silver staining for increased sensitivity.
     • Activity-based enzyme detection helps them to keep their biological purpose.
   • In their original form, bands show at different places in the gel matching the charge and molecular weight of the protein.
   • Protein measurements are approximated using a molecular weight marker as a guide.
   • Western blotting or in-gel activity tests allow one to examine proteins split by Native PAGE even further.
   • Detecting sensitivity and data accuracy have been raised by developments in imaging and quantification instruments using fluorescent colors and high-resolution scanners.

SDS PAGE vs. Native PAGE

Aspect	SDS PAGE	Native PAGE
Gel Nature	Denatured gel	Non-denatured gel
Denaturation	SDS is used to denature proteins	No denaturation step required
Separation Principle	Based on protein mass	Based on protein size and charge
Protein Stability	Proteins are not stable and cannot be recovered	Proteins are stable and can be recovered
Protein Conformation	Proteins are unfolded	Proteins retain their native conformation
Protein Activity	Protein activity is lost	Protein activity is preserved
Sample Preparation	Requires boiling in SDS sample buffer	Minimal or no sample preparation required
Resolution	Higher resolution due to denaturation	Lower resolution due to native conformation
Applications	Analysis of protein molecular weight and purity	Analysis of protein complexes and native structures

Applications of Native Polyacrylamide Gel Electrophoresis (PAGE)

In molecular biology and biochemistry, polyacrylamide gel electrophoresis (PAGE) is an extensively used method for the macromolecule separation and analysis, especially for proteins and nucleic acids. Its uses cover:

• Determining Molecular Weight- PAGE compares the migration of proteins over the gel to that of known molecular weight markers thereby enabling the estimate of their molecular weight.
• Assessing Purity– Separating elements of a sample allows PAGE to evaluate the purity of proteins or nucleic acids, therefore spotting contaminants or verifying the presence of a single species.
• Protein Quantification– By means of analysis of the intensity of stained bands matching to the proteins of interest, the method helps to quantify proteins.
• Monitoring Protein Integrity– Monitoring variations in protein composition in bodily fluids using PAGE can be rather important for diagnosis-related needs.
• Peptide Mapping– It helps peptide mapping, which is necessary for protein characterisation and fragment identification by means of which particular protein fragments are identified.
• Analyzing Protein Subunits and Aggregation– PAGE helps to identify aggregation states and ascertain the component makeup of proteins, therefore offering understanding of protein structure and function.
• Comparative Analysis- The method helps to compare polypeptide compositions between several samples, therefore supporting research on protein expression and variation.
• Western Blotting– Often used as a preparation step for Western blotting, PAGE is a process wherein separated proteins are placed on membranes for the purpose of specifically antibody detection.

Advantages of Native Polyacrylamide Gel Electrophoresis (PAGE)

For the study of proteins in their non-denatured forms, Native Polyacrylamide Gel Electrophoresis (PAGE) has a number of benefits.

• Preserves Protein Function– Maintains natural structure and activity.
• Studies Protein Complexes– Keeps non-covalent interactions intact.
• Separation by Multiple Factors– Uses size, shape, and charge for analysis.
• Protein Integrity– Allows protein recovery for further studies.

Disadvantages of Native Polyacrylamide Gel Electrophoresis (PAGE)

• Complex Interpretation– Separation depends on size, shape, and charge, making results harder to interpret.
• Low Resolution– Proteins with similar charge-to-mass ratios may not separate well.
• Sensitive to Conditions– pH and temperature can affect protein migration.
• Protein Aggregation– Some proteins may form aggregates, affecting separation.

Agarose vs polyacrylamide gel electrophoresis

Aspect	Agarose Gel Electrophoresis	Polyacrylamide Gel Electrophoresis
Composition	Natural polysaccharide extracted from seaweed.	Synthetic polymer formed from acrylamide monomers.
Pore Size	Larger pores suitable for separating large DNA fragments.	Smaller pores ideal for resolving proteins and small nucleic acids.
Applications	Commonly used for DNA and RNA analysis, including fragment sizing and purification.	Primarily used for protein analysis and small nucleic acid fragments, such as in SDS-PAGE.
Resolution	Lower resolution; less effective for distinguishing molecules with small size differences.	Higher resolution; capable of separating molecules with minor size variations.
Preparation	Easier to prepare; gels are typically cast horizontally and can be remelted and reused.	Requires careful handling due to toxicity; gels are usually cast vertically and cannot be reused.
Toxicity	Considered non-toxic and safer to handle.	Acrylamide is a neurotoxin; proper protective measures are necessary during preparation and handling.
Cost	Generally less expensive, making it suitable for routine analyses.	More costly due to the complexity of preparation and materials involved.

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