Molecular Techniques and Methods

Native Gel Electrophoresis

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Under native conditions, separation of proteins depends on many factors including size, shape, and native charge. One straightforward approach to native gel electrophoresis is to leave out the SDS and reducing agent (DTT) from the standard SDS-PAGE. The gel and electrohpresis solutions are prepared without SDS.

"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size. The electric charge driving the electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. This charge will, of course, depend on the amino acid composition of the protein as well as post-translational modifications such as addition of sialic acids. Since the protein retains its folded conformation, its hydrodynamic size and mobility on the gel will also vary with the nature of this conformation (higher mobility for more compact conformations, lower for larger structures like oligomers). If native PAGE is carried out near neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation, self-association or aggregation, and the binding of other proteins or compounds. Thus native gels can be sensitive to any process that alters either the charge or the conformation of a protein. This makes them excellent tools for detecting things such as: changes in charge due to chemical degradation (e.g. deamidation) unfolded, "molten globule", or other modified conformations oligomers and aggregates (both covalent and non-covalent) binding events (protein-protein or protein-ligand) These properties, and their relatively high throughput, make native gels excellent tools for analyzing accelerated stability samples, demonstrating comparability of different lots or processes, or examining the effects of excipients. Another advantage of native gels is that it is possible to recover proteins in their native state after the separation. Recovery of active biological materials may, however, need to be done prior to any fixing or staining.




MATERIALS AND SOLUTIONS

40% Acrylamide:Bis Solution (37.5:1)
Acrylamide ----------------------------------------------- 100 g
Bis-acrylamide -------------------------------------------- 2.65 g
  • Add deionized H2O to make a final volume of ----------- 250 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    4 x Separating Gel Buffer (200 ml)
    Tris (FW=121.1) ---------------------------------------- 36.3 g
    Deionized H2O ------------------------------------------ 150 ml
  • Adjust the pH to 8.8 with HCl.
    Add deionized H2O to make a final volume of ---------- 200 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    4 x Stacking Gel Buffer (50 ml)
    Tris (FW=121.1) ---------------------------------------- 15.1 g
    Deionized H2O ------------------------------------------ 40 ml
  • Adjust the pH to 6.8 with HCl.
  • Add deionized H2O to make a final volume of ---------- 50 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    10% Ammonium Persulfate (10 ml)
    Ammonium persulfate ----------------------------------- 1.0 g
    Deionized H2O ----------------------------------------- 10 ml
  • Keep in a brown glass bottle at 4oC.


    Electrophoresis Buffer (2 liter)
    0.192 M Glycine --------------------------------------- 28.8 g
    0.025 M Tris (FW=121.1) ----------------------------- 6.0 g
    Add deionized H2O to make a final volume of --------- 2 liter
  • The solution should be at about pH 8.3 without adjustment.
  • This solution is readily made fresh each time.


    H2O-Saturated n-Butanol (55 ml)
    n-Butanol ---------------------------------------------- 50 ml
    Deionized H2O ----------------------------------------- 5 ml
  • Combine in a bottle and shake. Use the top phase to overlay gels.
  • Store at room temperature indefinitely.


    2 x Sample Buffer (10 ml)
    4 X Stacking Gel Buffer (pH 6.8) ---------------------- 2.5 ml
    20% Glycerol ------------------------------------------ 2 ml
    0.02% Bromophenol Blue ------------------------------ 40 ul of 5% Bromophenol Blue
    Deionized H2O ----------------------------------------- 5.5 ml
  • Store 0.5 ml aliquots at -20oC for 6 months.


    Staining Solution (250 ml)
    Coomassie Brilliant Blue R250 -------------------------- 0.25 g
    (or, PAGE Blue 83 -------------------------------------- 0.25 g)
    Methanol ----------------------------------------------- 125 ml
    Glacial Acetic acid -------------------------------------- 25 ml
    Deionized H2O ----------------------------------------- 100 ml
  • Dissolve the Coomassie dye in the methanol component first, then add the acid and water.
  • If dissolved in a different order, the dye's staining behavior may differ. Use when freshly made.


    Destaining Solution (1 liter)
    Methanol ----------------------------------------------- 100 ml
    Glacial Acetic acid -------------------------------------- 100 ml
    Deionized H2O ----------------------------------------- 800 ml
  • Mix thoroughly. Use when freshly made.




    PROCEDURES

    1. Thoroughly clean and dry the glass plates and three spacers, then assemble them with bulldog clips. Clamp the chamber in an upright, level position.

    2. Prepare 10 ml Separating Gel Mixture as follows.

    4%
    5%
    6%
    8%
    10%
    40% Acrylamide:Bis Solution (37.5:1)
    1 ml
    1.25 ml
    1.5 ml
    2 ml
    2.5 ml
    4 x Separating Gel Buffer
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml
    50% Glycerol
    2.5 ml
    -
    -
    -
    -
    ddH2O
    4 ml
    6.25 ml
    6 ml
    5.5 ml
    5 ml


    3. Degas the solution, and then add:
    10% Ammonium Persulfate -------------------------------- 50 ul
    TEMED (N, N, N', N'-tetramethyl-ethylenediamine) ------------- 10 ul

    4. Mix gently and use
    immediately (because polymerization starts when the TEMED is added). Carefully pour the freshly mixed solution into the chamber without generating air bubbles. Pour to a level about 1 cm below where the bottom of the well-forming comb will come when it is in position.

    5. Carefully overlayer the acrylamide solution with H2O-saturated n-butanol without mixing to eliminate oxygen and generate a flat top to the gel.

    6. Polymerize the acrylamide for 1 hour.

    7. Prepare the 4 ml Stacking Gel Solution as follows. Mix the following:

    40% Acrylamide:Bis Solution (37.5:1)
    0.4 ml
    4 x Stacking Gel Buffer
    1.0 ml
    ddH2O
    2.6 ml


    8. Degas the Stacking Gel Solution, then add:
    10% Ammonium Persulfate -------------------------------- 20 ul
    TEMED ---------------------------------------------- 5 ul

    9. Mix gently and use
    immediately.
    Pour off the n-butanol from the polymerized Separating Gel, wash the gel top with water, and fill the gap remaining in the chamber with the Stacking Gel mixture. Insert the comb.

    10. Polymerize the acrylamide for 1 hour.

    11. When the Stacking Gel has polymerized, remove the comb without distorting the shapes of the well. Remove the clips holding the plates together, and install the gel in the apparatus.

    12. Fill apparatus with Reservoir Buffer. Push out the bottom spacer from the gel and remove bubbles from both the top and underneath of the gel. Use the gel immediately.

    13. While the gel is polymerizing, prepare samples for electrophoresis.
    Dissolve the protein sample solution in a same volume of 2 X Sample Buffer, or dissolve a dry sample in 1 X Sample Buffer. The concentration of sample in the solution should be such as to give a sufficient amount of protein in a volume not greater than the size of the sample well.
    (The bromophenol blue dye in Sample Buffer indicates when the sample solution is acidic by turning yellow. If this happens, add a little NaOH, enough to just turn the color
    blue.)

    14. Load the gel with 10-30 ul (20-50 ug) Protein Sample Solution by pipet.
  • 20 ug for PCDF memebrane blotting
  • 50 ug for nitrocellulose blotting

    15. Start electrophoresis immediately by turning on power. On a gel of 1 mm thickness and 15 cm length, an applied voltage of about 150 volts gives a current of about 20 mA (falling during electrophoresis if constant voltage is employed). The bromophenol blue dye front takes about 3 hours to reach the bottom of the gel. Greater voltage speeds up electrophoresis, but generates more heat in the gel.

    16. Remove the gel from between the glass plates.

    17. Stain the gel in the Staining Solution for 2-3 hours.

    18. Remove the dye that is not bound to protein in Destaining Solution. After about 24 hours, with gentle agitation and several changes of Destaining Solution, the gel background becomes colorless and leaves protein bands colored blue, purple, or red. Coomassie Brilliant Blue R250 and PAGE Blue 83 each visibly stain as little as 0.1-1 ug of protein in a band of about 1 cm width.




    NOTES

  • The glycerol in the 2 X Sample Buffer increases the density of the sample, to aid the loading of it onto the gel.


  • The bromophenol blue dye in the 2 X Sample Buffer aids loading of the sample, by making it visible, and indicates the position of the front of electrophoresis in the gel. The bromophenol blue also indicates when the sample solution is acidic by turning yellow. If this happens, add a little NaOH, enough to just turn the color blue.


  • The degassing stage removes oxygen, which inhibits polymerization by virtue of mopping up free radicals, and also discourages bubble formation when pouring the gel.


  • The polymerization of acrylamide and bisacrylamide is initiated by the addition of TEMED and ammonium persulfate. The ammonium persulfate activates the TEMED and leaves it with an unpaired electron. This radical reacts with an acrylamide monomer to produce a new radical that reacts with another monomer, and so on to build up a polymer. The bis acrylamide is incorporated into polymer chains this way and so forms crosslinks between them.





  • KIT INFORMATION




    REFERENCES

  • Deyl, Z., 1979, Electrophoresis. A survey of techniques and applications. Part A: Techniques. Elesevier, Amsterdam.


  • Hames, B.D., 1990, One-dimensional polyacrylamide gel electrophoresis. In "Gel Electrophoresis of Proteins. A Practical Approach (Hames, B.D. and Rickwood, D., eds.), pp.1-147. Oxford University Press, New York.


  • Laemmli, U.K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.


  • Studier, F.W., 1973, Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".



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