Native Polyacrylamide Gel Electrophoresis (PAGE)

What is Native Polyacrylamide Gel Electrophoresis?

Native Polyacrylamide Gel Electrophoresis (often written as Native PAGE) can be defined as a technique where proteins’ mixtures are separated under non-denaturing condition’s, so their natural Shape and Charge are mostly preserved.

In this method the protein’s are moved through a polyacrylamide matrix (PAM) by an electric field,and their mobility depends on Size/charge ratio , and also on the native Conformation which sometimes dominates the migration.

It is important to note that the samples are not treated with SDS, so the proteins keep their tertiary structures, And this makes the method useful for studying Enzyme activities or Protein/Protein complexes etc.

The gel’s running buffer is maintained at a controlled pH (around 8.3 usually) but sometimes it shift’s because of the ionic gradients, creating slight differences that affect the migration of the native proteins, it seems unavoidable in practical work.

This approach is considered a key component in biochemical labs because it gives a look into the functional State of many proteins, and run-on steps often occur when proteins aggregate in to bigger complexes during the run, which influence the bands.

In general terms, Native PAGE occurs without the reducing agent’s (DTT/β-ME) and without boiling, so the preserved Quaternary Structure become important for interpreting the band pattern’s.

The methodology of polyacrylamide gel electrophoresis (PAGE) emerged in the late 1950s when researchers began to use cross-linked polyacrylamide gels as a supporting matrix for zone electrophoresis of proteins and nucleic acids.Early work in 1959 reported acrylamide gel as a supporting medium for zone electrophoresis.

Over subsequent decades the technique was refined—especially via the discontinuous electrophoresis system developed in the 1960s (by Ornstein and Davis) which improved resolution by stacking and resolving gel zones.

The variant of PAGE in which proteins remain in their native (non-denatured) conformation came to be referred to as “native PAGE” and contrasts with the denaturing form (e.g., SDS-PAGE) that became widely used after the landmark work of Ulrich K. Laemmli in 1970.As electrophoretic and biochemical methods matured, native PAGE found its niche in analysing protein complexes, their oligomeric state and functional forms, rather than simply molecular weight.

In more recent years further innovations such as the “blue native PAGE” (BN-PAGE) and high-resolution clear native (hrCN-PAGE) techniques have extended the native gel concept and improved resolution of intact protein assemblies.

Thus, the history of native PAGE reflects a broader evolution from simple gel media and zone electrophoresis to sophisticated tools for studying intact biomolecular structures and complexes in near-native states.

Principle of Native Polyacrylamide Gel Electrophoresis (PAGE)

The basic principle of Native PAGE can be defined as separation of proteins’ in their natural Conformation’s through a polyacrylamide matrix under an applied electric field, maintaining both Shape and Charge during migration.

In this technique the proteins are moved according to their intrinsic charge-to-mass ratio, and also the 3-D Structure influences the path through the gel which create’s a slightly unpredictable mobility pattern.

The electric field drives molecules from the stacking zone in to the resolving gel, where pore size and buffer pH (≈8.3 sometimes written 8.3 ) determine how quickly the native Complexes travel.

It is worth mentioning that no SDS or reducing agent’s are added, so the Protein/Protein interactions are preserved, And this allow’s the observation of oligomers, enzyme’s or other functional states.

Proteins with higher net negative charge move faster toward the anode, but bulky or folded domains slow them down, creating a sturdy and hardy balance between Charge/Size/Shape factors.

The mobility is influenced by the native buffer system (Tris-Gly or related) which also gives a look into the electro-osmotic properties of the gel, leading to slight run-on behavior when complexes aggregate, it happen’s often.

Overall it can be stated that Native PAGE relies on preserving biological Activity, keeping proteins in nearly physiological structure’s, so the separation reflect conformation rather than only molecular mass etc.

Types of Native Polyacrylamide Gel Electrophoresis

1. Blue Native PAGE (BN-PAGE) – This technique is used for separation of large Protein Complexe’s in their native conformation, and it depends on Coomassie Blue G-250 dye which attach to the proteins’ surface and create a charge shift,also the mobility often get influenced by the detergent/protein ratio that scientist’s chooses.

2. Clear Native PAGE (CN-PAGE) – This method is performed without Coomassie dye, it is important to note that proteins migrates depending mainly on their own charge–to–mass ratio,there is no artificial charge shift, so the resolution can be slightly different and sometimes harder to interpret but it gives a look into the “true” mobility of some Enzyme’s.

3. High-Resolution Clear Native PAGE (hrCN-PAGE) – A modified CN approach, where optimized buffer’s and gradient gel’s are used to achieve better separation of membrane complexes,in some cases the ampholyte content is raised which perhaps gives more distinct Bands, also the protocol often use 4–14 % gradients etc.

4. Blue-Native / SDS-PAGE Two-Dimensional System– This 2-D combination is used when the researcher need’s to first keep complexes intact (BN step) and then separate subunits on SDS gel, it is considered a key approach in studying respiratory Chain Complexes, and it sometime reveal subunit stoichiometry even if the sample is partially unstable.

5. Native Isozymes PAGE– This technique is used for analyzing Isoenzyme pattern’s (like Peroxidase or Esterase etc.) where enzyme activities are retained, bands are visualized by activity stains (e.g. substrate-based; reaction forms colored precipitate),And then different organ/tissue pattern can be compared.

6 . Charge-Shift Native PAG– A variant where small anionic or cationic additives (i.e. imidazole) are applied to modify migration behavior, proteins’ charge may be adjusted in to narrow ranges, this often help with complexes that normally co-migrate.

7. Gradient Native PAGE– The gel is cast with increasing % acrylamide (4–20 %), and this allow larger assemblies to enter easily while smaller ones resolve sharply deeper in the matrix, movement also affected by electric field strength which researcher’s adjust depending on sample sensitivity.

8. Detergent Native PAGE– Uses mild detergents (e.g. digitonin / DDM) under native condition’s to keep membrane protein’s soluble, the technique has been shown that it can preserve supramolecular assemblies although stability varies,And some run-on separation occurs because detergent micelles changes apparent mass.

Requirements for Polyacrylamide Gel Electrophoresis (PAGE)

• A properly prepared acrylamide / bis-acrylamide solution is needed, and the ratio often decides pore sizes,Also polymerization sometimes behave slightly different when temperature changes.
• he gel must contains initiators like APS (ammonium persulfate) and TEMED which catalyze Free-Radical formation,these components’ amounts are adjusted carefully because too much APS create brittle gel’s.
• A continuous or discontinuous buffer system is required (e.g. Tris-Glycine) , and the ions gives the conductivity that actually let’s the proteins’ migrate in to the gel matrix.
• A sample loading dye is used to provide tracking and density, however in PAGE (native or SDS) the Dye’s composition varies, and sometimes glycerol % change the sample sinking behavior.
• The electrophoresis apparatus need’s stable power supply, because current fluctuations influence Band resolutions,It is well known that 120 V run’s can overheat if cooling not applied.
• An appropriate protein sample, prepared with or without SDS depending on native/SDS mode, is required, proteins sometimes aggregates during preparation,And this reduce migration.
• Running conditions (voltage, time, buffer temperature 12 °C or 20°C etc.) must be controlled, and slight overheating produce “smiling” effect in the lanes.
• Staining solution’s (Coomassie, silver stain etc.) are needed to visualize the separated proteins,they provide contrast but also can cause Background if washing steps are skipped.
• Clean glass-plate assembly and spacers are required for casting the gel, uneven alignment often leads to slanted Bands which is annoying for quantification.
• A suitable protein ladder/marker is also required for estimating molecular weight’s, though sometimes the marker behaves differently in in-vitro vs in vitro conditions.

Procedure of Native Polyacrylamide Gel Electrophoresis

• Prepare the protein sample in a non-denaturing buffer that preserves physiological pH and ionic strength, and ensure no SDS or urea are present because native conformation must be retained.
• Clarify or centrifuge the sample to remove particulate matter, protein concentration is measured (e.g. by Bradford) and adjusted so that roughly 0.1–2 µg/µL is loaded depending on detection method.
• Add a non-denaturing loading dye (contains tracking dye like bromophenol blue and glycerol for density), the dye’s amount is kept small to avoid charge perturbation.
• Prepare acrylamide/bis-acrylamide solution at the chosen % (for example 5–7 % for high-MW, 10–15 % for smaller proteins), gradient gels are cast if complex mixtures are expected.
• Degas the gel solution briefly if available, then add APS (ammonium persulfate) and TEMED to initiate polymerization, the reagents’ volumes are kept minimal and mixed quickly.
• Pour the resolving gel between clean glass plates, insert spacer’s and place a comb to form wells, allow polymerization to complete at room temperature or slightly cooler.
• Remove the comb carefully after polymerization and rinse the wells with running buffer, any bubbles or irregular wells are noted and gently removed.
• Assemble the gel in the electrophoresis tank and fill the upper and lower reservoirs with native running buffer (e.g. Tris–Glycine), the buffer should be degassed and at the right ionic strength.
• Pre-run the gel briefly at low voltage if desired,to equilibrate the gel and remove residual unpolymerized acrylamide etc., this is optional but sometimes recommended.
• Load the molecular weight and/or native mobility marker in one lane, then load the prepared sample’s into wells using a fine pipette tip, avoid touching the bottom of the wells to prevent leakage.
• Run the gel at a constant voltage or current (typical 50–150 V depending on gel size and apparatus) while controlling temperature (ice or cooling unit if needed) to prevent overheating and band distortion.
• Monitor the migration by tracking dye, run until satisfactory separation is achieved, stopping earlier if activity-assays will be performed in-gel.
• Stop the run and disassemble the gel plates carefully, the gel is rinsed briefly in buffer to remove salts if immediate staining is planned.
• Stain the gel with Coomassie Brilliant Blue for general protein detection or use silver stain for higher sensitivity, activity stains (substrate-based) are employed when native enzymatic function must be observed.
• Destain (for Coomassie) or develop (for silver/activity) until background is acceptable, and then document bands using a scanner or imaging system, fluorescent stains may be used for more sensitive quantification.
• Estimate protein apparent mass/mobility using the marker, and, if needed, excise bands for further analysis (e.g., in-gel digestion, western blot after gentle transfer), note that transfer conditions differ from SDS-PAGE and must be optimized.

SDS PAGE vs. Native PAGE

Applications of Native Polyacrylamide Gel Electrophoresis (PAGE)

• It is used for separation of protein’s in their native folded State because the mobility reflect’s the protein/ lipid ratio and the charge in a more physiological way, which gives a look into the reaction of complexes.
• This allows protein complex’s to be isolated for later assay’s (enzyme activity,etc.), and sometimes the folded subunits are kept intact, it is important to note that this helps with stability studies also.
• They are applied for checking Oligomeric state’s of Proteins, And this method gives approximate size distribution even when detergent in-vitro conditions are avoided.
• These are used for observing Protein–Protein interaction’s, the band shift is taken as evidence, however sometimes the migration are influenced by buffer ionic strength ,which scientist’s must adjust.
• It provides a way for analyzing conformational transitions (e.g., heat-induced unfolding), and the different shapes are separated because the Shape contribute’s to mobility, it seems a little indirect but commonly used.
• This is used for verifying sample purity prior to MS (mass spectrometry) or chromatography / electrophoresis workflows, also the faint Band’s indicate contaminant’s.
• They support study on membrane-associated proteins’ that maintain partial “native” conformations, even though the Gel system is not fully physiological.
• These are used in routine QC of enzyme prep’s, sometimes the pattern tells about the lost Activity in to storage.
• It helps in characterizing isoenzyme’s (i.e. same enzyme but different charge forms), and the Run-on pattern indicates tissue origin or developmental Stage.
• This can separate nucleic acid-binding proteins without denaturation, furthermore,it can be said that the shift assay (DNA/protein) becomes more Sensitive on native PAGE.
• They are applied for antibody screening, the Immunoglobulin’s show unique migration due to their bulky shape, and misfolded form’s are sometimes detected inadvertently.
• These allow recovery of intact proteins from the gel for downstream assays like activity testing, refolding attempts, or immunization experiment’s.

Advantages of Native Polyacrylamide Gel Electrophoresis (PAGE)

• It preserves protein’s native folding and Activity which is important to note that this helps in studying functional complexes, also the migration reflect’s real charge/shape.
• This allows separation without harsh denaturant’s ,and this give a more Physiological Pattern, sometimes the mobility is clearer than SDS–PAGE.
• They permit analysis of Protein–Protein interaction’s because complexes stay intact, the band shift’s provide useful evidence, it is well known that such assays are widely used.
• These offer recovery of intact proteins from Gel for downstream test’s (enzyme activity, MS), and the sample purity often remain’s higher.
• It supports characterization of isoenzyme’s and charge variants, the Technique show’s subtle differences that SDS systems hide.
• This reduces risk of artifact’s caused by unfolding, and in general terms the method have lower chemical disturbance on proteins.
• They accommodate membrane-associated protein’s that retain partial conformation’s, although buffer strength sometimes need adjustment, the approach still remain’s sturdy and hardy.

Limitations of Native Polyacrylamide Gel Electrophoresis (PAGE)

• It can give ambiguous mobility because the Shape and Charge of protein’s vary unpredictably, and it is worth mentioning that migration does not directly equal molecular mass in many cases.
• This often require’s very careful buffer control, ionic strength change’s the patterns, And small fluctuations produce confusing band’s.
• They offer lower resolution for closely sized proteins’, the folded State hide’s size differences which SDS–PAGE usually separate’s much better.
• These are sensitive to sample purity ,and contaminant’s may form mixed complexes that shift the mobility, moreover the interpretation become’s messy.
• It is not ideal for membrane-protein’s that need detergent; too much detergent disrupt’s “native” Conditions, too little makes aggregation.
• This complicates quantification because Staining efficiency depend’s on conformation, it is well known that some folded domain’s stain poorly, giving uneven intensity’s.
• They provide limited denaturation control, so misfolded forms might run similar to Correct conformations, creating comma-splice issues in data evaluation.
• These usually take longer time for optimization (buffer pH, acrylamide %, ampholyte’s etc.), and the method get’s affected by temperature shift’s (12 °C vs 12°C) in unpredictable way’s.

Agarose vs polyacrylamide gel electrophoresis

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