Introduction to PAGE – Separation of Proteins Based on Size

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Polyacrylamide gels are formed by the reaction of acrylamide and bis-acrylamide (N,N’-methylenebisacrylamide) that results in highly cross-linked gel matrix. The gel acts as a sieve through which the proteins move in response to the electric field. Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge. By denaturing the proteins and giving them a uniform negative charge, it is possible to separate them based on the size as they migrate towards the positive electrode.

SDS-PAGE of protein samples and color burst protein marker

Figure 1: SDS-PAGE of protein samples and color burst protein marker (C1992)


Protocol

Materials and reagents required

  • Vertical electrophoresis chamber with power supply, glass plates, spacers and combs

  • Acrylamide/bis-acrylamide solution (A3449, A3574, A3699)
    OR prepare 30% acrylamide solution using the following reagents:

         Acrylamide (01696) 29.2 g
         Bis-acrylamide (14602) 0.8 g
         Distilled water 100 mL


  • Gel casting buffers: For discontinuous gels, the buffers required to prepare resolving gel and stacking gel are different.

         1.5 M Tris-HCl, pH 8.8 (to prepare resolving gel): Dissolve 18.15 g of Tris base in 80 mL distilled water. Adjust pH to
         8.8 using 6N HCl. Make up the final volume to 100 mL with distilled water.

         0.5 M Tris-HCl, pH 6.8 (to prepare stacking gel): Dissolve 6 g of Tris base in 80 mL distilled water. Adjust pH to 6.8
         using 6N HCl. Make up the final volume to 100 mL with distilled water.


  • 2X Laemmli loading buffer:

         Bromophenol blue (B5525) 0.004%
         2-mercaptoethanol 10%
         Glycerol 20%
         SDS (L3771) 4%
         Tris-HCl (93362) 0.125 M

  • 10X Running buffer (GE28-9902-52; acts as both anode and cathode buffer)
    OR prepare 1X running buffer using the following reagents:

         Tris-HCl (93362) 25 mM
         Glycine (G8898) 200 mM
         SDS (L3771) 0.1% (w/v)

  • 10% SDS (L3771)

  • 10% Ammonium persulphate (A3678)

  • TEMED (T9281)

NOTE: Acrylamide and bis-acrylamide are neurotoxic in nature. All the steps should be performed wearing powder-free gloves.


Gel preparation

  • Clean the glass plates and spacers of the gel casting unit with deionized water and ethanol.
  • Assemble the plates with the spacers on a stable, even surface.
  • Prepare resolving gel solution using the following volumes (for 10 mL) depending on the percentage of gel required.
Gel % Water (mL) 30% acrylamide (mL) 1.5 M Tris-HCl, pH 8.8 (mL) 10% SDS (µL) 10% APS (µL) TEMED*(µL)
8% 4.6 2.6 2.6 100 100 10
10% 3.8 3.4 2.6 100 100 10
12% 3.2 4.0 2.6 100 100 10
15% 2.2 5.0 2.6 100 100 10

*TEMED must be the last ingredient added

  • Pour the gel solution in the plates assembled with spacers. To maintain an even and horizontal resolving gel surface, layer the surface with water or isopropanol (I9516).

  • Allow the gel to set for about 20-30 min at room temperature.

  • Prepare stacking gel solution using the following volumes (for 10 mL):
Gel % Water (mL) 30% acrylamide (mL) 0.5 M Tris-HCl, pH 6.8 (mL) 10% SDS (µL) 10% APS (µL) TEMED*(µL)
5% 5.86
1.34
2.6 100 100 10

*TEMED must be the last ingredient added

  • Discard the water on the resolving gel.

  • Add the 5% stacking gel solution until it overflows. Insert the comb immediately ensuring no air bubbles are trapped in the gel or near the wells.

  • Allow the gel to set for about 20-30 min at room temperature.


Sample preparation

  • To a volume of protein sample (cell or tissue lysate), add equal volume of loading buffer.

  • Boil the above mixture at 95°C for 5 min. Centrifuge at 16000G for 5 min.

  • These samples can be stored at -20°C or may be used to proceed with gel electrophoresis.


Gel staining

Coomassie Blue staining: Staining of protein gels with Coomassie Brilliant Blue R-250 is a common procedure to visualize proteins resolved by SDS-PAGE. It is highly sensitive and is suitable for long-term storage of the gels.


Reagents required


Procedure

  • After the electrophoresis, place the gel in a plastic tray containing gel fix solution. Place the tray on a rocking table and fix the proteins for 2 hours.

  • Remove the gel fix solution and add Coomassie solution.  Place on a rocking table and stain the gel for 2-4 hours.

  • After the staining step, wash the gel several times with distilled water to remove excess stain.

  • Add destain solution to the gel. Place on rocking table and destain for about 4 hours till clear blue bands on clear background are visible.

  • After destaining, the gels may be stored in gel storage solution and photographed as required.


Silver staining

Sigma-Aldrich offers a highly sensitive protein detection silver stain kit suitable for SDS-PAGE gels. The following reagents are additionally needed:

  • Fixing solution

         Ethanol (E7023) 50 mL
         Acetic acid 10 mL
         Water 40 mL

  • 30% ethanol (E7023) solution


Procedure

  • After electrophoretic run immerse the gel in fixing solution for 40 min. An overnight immersion in fixer will produce clearer background.

  • Remove the fixing solution and wash the gel for 10 min with 30% ethanol solution followed by wash with ultrapure water for 10 min.

  • Decant the water and incubate the gel in sensitizer solution for 10 min.

  • Remove the sensitizer solution and wash the gel twice with water, each wash lasting for 10 min.

  • Decant the water and immerse the gel for 10 min in silver solution.

  • Decant silver solution and wash the gel in water for 1.5 min.

  • Discard the water and immerse the gel in developer solution for 3 to 7 min.

  • Add 5 mL of stop solution to the developer solution and incubate for 5 min. Remove the developer/stop solution and wash the gel in ultrapure water for 15 min.

  • The gel can be photographed and also stored in fresh, ultrapure water.

For double staining, stain the gel using CBB R-250 followed by silver stain using the procedures above.

Fluorescent Stains: Sigma-Aldrich offers fluorescent SYPRO and Lucy stains for protein electrophoresis. The gels can be either immersed in the fluorescent stain in dark or the gel can be mixed with cathode buffer during the electrophoresis.

 

Product No. Name
S4942 SYPRO® Ruby Protein Gel Stain
S5692 SYPRO® Orange Protein Gel Stain
68721 LUCY® 506 solution
43772 LUCY® 565 solution
41629 LUCY® 569 solution
51723 LUCY® 565 Molecular Weight Standard Kit
51153 LUCY® Starter Kit


The staining protocols will depend on the stain being used and may be found here:


Reversible gel staining

Reversible gel stains allows the user to proceed to western blotting of proteins after SDS-PAGE.

R-PROB staining: R-PROB is a unique stain that detects proteins on PAGE gels and western blots.

Reagents required:


Procedure

  • Immerse the gels post-electrophoresis in fixing solution for 20 mins. Repeat this step twice.

  • Rinse the gel in water twice, each wash lasting 30 mins.

  • Incubate the gel in staining solution for 20-40 mins with gentle agitation.

  • Wash off excess stain in 10% acetic acid.

  • For destaining, wash the gel with EDTA followed by two washes of 15 mins each in water or fixing solution.

Copper staining: To confirm the migration and separation of proteins, the gel may be stained with a reversible stain such as CuCl2.


Reagents required

  • 0.3 M CuCl2 solution

  • 0.25 M Tris and 0.25 M EDTA solution


Procedure

  • Rinse the gels post-electrophoresis in distilled water for a maximum of 30 min.

  • Immerse the gel in 0.3 M CuCl2 solution for 10 min.

  • Rinse with de-ionized water.

  • The proteins can be visualized as clear zones in a blue background.

  • For destaining, wash the stained gels in 0.25 M Tris and 0.25 M EDTA solution, pH 9, repeatedly.

  • Move the destained gel to transfer buffer before proceeding with the transfer setup.

 

Sigma-Aldrich Blotting and Vertical Electrophoresis System

Figure 2: Sigma-Aldrich Blotting and Vertical Electrophoresis System


If the transfer of proteins to a membrane after electrophoresis is desired, please find the protocol here.

 Reference

  • Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular cloning: a laboratory manual. New York: Cold spring harbor laboratory press, 1989.

 

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