Molecular Techniques and Methods

Two-Dimensional Polyacrylamide Gel Electrophoresis

Copy Right © 2001/ Institute of Molecular Development LLC


INTRODUCTION

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) developed by O'Farrell (1975) employs separation of denatured proteins according to two different parameters, isoelectric point (IEF) and molecular weight (MW).
Individual eukaryotic cells may contain 10,000 polypeptides with varying abundancies. However, by 2-D PAGE analysis only about 2,000 individual proteins have so far been detected. This is usually interpreted as the inability to detect minor proteins, but Duncan and McConkey (1982) have argued that in fact 2,000 is close to the number of proteins in a cell and that the remaining rare mRNAs are rarely, if ever translated. If this is the case, then 2-D PAGE represents an even more powerful technique than previously expected for investigating changes in cellular physiology.
2-D PAGE system also detects proteins that contain single amino acid substitutions, which confer a change in isoelectric point on the protein. This has resulted in definitive identification of missense mutations within proteins and the visualization of mistranslated proteins.

Isoelectric points
Differences in proteins isoelectric points are the basis of separations by isoelectric focusing (IEF). The pI is the pH at which a protein will not migrate in an electric field and is determined by the charged groups in the protein. Proteins can carry positive, negative or zero charge depending on their local pH, and for every protein there is a specific pH at which its net charge is zero; this is its pI. pI's generally fall in the range 3 - 12, with most being around 4 - 7. When a protein is placed in a medium with a pH gradient and subjected to an electric field it will initially move towards the electrode with the opposite charge. During migration through the pH gradient the protein will pick up or lose protons. As it migrates the net charge and the mobility will decrease and the protein will slow down. Eventually the protein will arrive at the point in the pH gradient which is equal to its pI. Here it will be uncharged and hence stop migrating. If the protein should happen to diffuse to a region outside its pI it will pick up a charge and hence move back to the position where it is neutral. In this way proteins are condensed, or focused, into sharp bands.

pH gradients
pH gradients are established by using carrier ampholytes. These are molecules which are engineered to have a particular pK. By mixing them it is possible to form a mixture which will establish a pH gradient. Originally these molecules were cast into the gel and an electric field applied to form the pH gradient. This was an efficient method but suffered because the ampholytes would drift during the isoelectric focusing step. It is also extremely difficult to reproduce gradients in these systems. Nowadays immobilized pH gradients (IPG's) are used. The carrier ampholytes are attached to acrylamide molecules and cast into the gels to form a pH gradient that is fixed. This prevents drift in the gel and also ensures that they can be cast in an efficient and reproducible manner.




MATERIALS AND SOLUTIONS

One-Dimensional Isoelectric Focusing Gel Electrophoresis

Sonication Buffer (1 ml)
10 mM Tris-HCl (pH 7.4) ---------------------------------------------- 10 ul of 1 M Tris-HCl
5 mM MgCl2 ---------------------------------------------------------- 5 ul of 1 M MgCl2
Deionized H2O -------------------------------------------------------- 985 ul

  • Store at 4oC.


    Pancreatic Ribonuclease (1 ml)
    Pancreatic Ribonuclease --------------------------------------------- 50 mg
    Sonication Buffer ---------------------------------------------------- 1 ml
  • Store at -20oC in aliquots.


    Deoxyribonuclease (1 ml)
    Deoxyribonuclease -------------------------------------------------- 1 mg
    Sonication Buffer ---------------------------------------------------- 1 ml
  • Store at -20oC in aliquots.


    Lysis Buffer (10 ml)
    9.5M Urea ---------------------------------------------------------- 5.7 g Urea
    2% (v/v) 50% Igepal CA-630 -------------------------------------- 0.4 ml of 50% 50% Igepal CA-630
    2% (v/v) Ampholines (40%) (1.6%, pH range 5-7) ---------------- 100 ul of 40% Ampholines (pH5-7)
    2% (v/v) Ampholines (40%) (0.4%, pH range 3-10) --------------- 500 ul of 40% Ampholines (pH3-10)
    5% (v/v) 2-mercaptoethanol ---------------------------------------- 0.5 ml of 2-mercaptoethanol
    Add deionized H2O to make a final volume of -------------------- 10 ml
  • Store at -70oC in 0.5 ml aliquots.
  • Use an aliquot once and discard the remainder.


    Sample Overlay Buffer (10 ml)
    8M Urea ------------------------------------------------------------ 4.81 g
    2% (v/v) Ampholines (40%) (1.6%, pH range 5-7) ----------------- 100 ul of 40% Ampholines (pH5-7)
    2% (v/v) Ampholines (40%) (0.4%, pH range 3-10) ---------------- 500 ul of 40% Ampholines (pH3-10)
  • Divide into aliquotes and store at -70oC.


    KOH -Ethanol Solution
    KOH ------------------------------------------------------------------ 0.4 g
    Ethanol ---------------------------------------------------------------- 20 ml


    30% Acrylamide:Bis Solution for IEF (19:1) (66.6 ml)
    Acrylamide (MW=71.08) -------------------------------------------- 19 g
    NN'-methylene-bisacrylamide (MW=154.2) -------------------------- 1 g
    Add deionized H2O to make a final volume of ----------------------- 66.6 ml
  • Store in a dark glass bottle at 4oC for 3 months.


    Anode (+) Electrode Solution (3 L) - Lower chamber
    0.01 M Phosphoric acid --------------------------------------------- 30 ml of 1 M Phosphoric acid
    Deionized H2O ------------------------------------------------------ 2970 ml
  • Degas the solution.


    Cathode (-) Electrode Solution (1 liter) - Upper chamber
    0.02M NaOH ------------------------------------------------------ 4 ml of 5 M NaOH
    Deionized H2O ----------------------------------------------------- 996 ml
  • Degas the solution.


    40% Ampholines
    Keep sterile and store at 4oC.


    10% Ammonium Persulfate (10 ml)
    Ammonium persulfate ----------------------------------------------- 1g
    Deionized H2O ----------------------------------------------------- 9 ml


    SDS Sample Buffer (100 ml)
    0.06M Tris-HCl (pH 6.8) ------------------------------------------ 6 ml of 1 M Tris-HCl
    2% w/v SDS ------------------------------------------------------- 20 ml of 10% SDS
    5% (v/v) 2-mercaptoethanol ---------------------------------------- 5 ml of 2-mercaptoethanol
    10% (v/v) Glycerol ------------------------------------------------- 20 ml of 50% Glycerol
    0.1% Bromophenol blue -------------------------------------------- 2 ml of 5% Bromophenol blue
    Deionized H2O ----------------------------------------------------- 47 ml




    Two-Dimensional SDS-Slab Gel Electrophoresis

    40% Acrylamide:Bis Solution for SDS-PAGE (37.5:1) (250 ml)
    Acrylamide (MW=71.08) --------------------------------------------- 100 g
    N,N'-methylene-bisacrylamide (MW=154.2) ------------------------- 2.7 g
    Add deionized H2O to make a final volume of ----------------------- 250 ml
  • Store in a dark glass bottle at 4oC for 3 months.


    30% Acrylamide:Bis Solution for SDS-PAGE (37.5:1) (250 ml)
    Acrylamide (MW=71.08) --------------------------------------------- 75 g
    N,N'-methylene-bisacrylamide (MW=154.2) ------------------------- 2 g
    Add deionized H2O to make a final volume of ----------------------- 250 ml
  • Store in a dark glass bottle at 4oC for 3 months.


    5 x Tris-Glycine-SDS Running Buffer (1 L)
    0.025 M Tris base ---------------------------------------------------- 15.1 g
    0.192 M Glycine ----------------------------------------------------- 94 g
    0.1 - 1% (w/v) SDS -------------------------------------------------- 10 - 100 ml of 10% SDS
    Add deionized H2O to make a final volume of ----------------------- 1 liter
  • Do not titrate this or add any salt.


    4x Separating Gel Buffer (200 ml)
    1.5M Tris-HCl (pH 8.8) ------------------------------------------ 150 ml of 2 M Tris-HCl
    0.4% (w/v) SDS -------------------------------------------------- 8 ml of 10% SDS
    Deionized H2O ---------------------------------------------------- 42 ml
  • Store at 4oC.


    4x Stacking Gel Buffer (200 ml)
    0.5M Tris-HCl (pH 6.8) ------------------------------------------- 100 ml of 1 M Tris-HCl
    0.4% (w/v) SDS --------------------------------------------------- 8 ml of 10% SDS
    Deionized H2O ----------------------------------------------------- 92 ml
  • Store at 4oC.


    Gel Fixer (100 ml)
    45% (v/v) Methanol ------------------------------------------------ 45 ml of 100% Methanol
    7.5% (v/v) Glacial acetic acid --------------------------------------- 7.5 ml of 100% Glacial acetic acid
    Deionized H2O ----------------------------------------------------- 47.5 ml


    Gel Staining Solution (100 ml)
    0.2% (w/v) Coomassie blue ---------------------------------------- 0.2 g
    45% (v/v) Methanol ------------------------------------------------ 45 ml of 100% Methanol
    10% (v/v) Glacial acetic acid --------------------------------------- 10 ml of 100% Glacial acetic acid
    Deionized H2O ----------------------------------------------------- 44.8 ml




    PROCEDURES

    Sample Preparation

    1. Pellet cells by centrifugation and resuspend in 100 ul Sonication Buffer at 4oC.

    2. Sonicate the cells with the microtip of a sonicator. Be very careful not to overheat the sample.

    3. Add 2 ul of RNase (50 mg/ml) and 2 ul of DNase (1 mg/ml) and incubate for 5 minutes at 4oC.

    4. Add solid urea to bring the sample to 9M (1 mg urea/ml of sample), which approximately doubles the volume.
  • Alternatively, lyophilize the protein and resuspend in 20-40 ul Lysis Buffer.

    5. Add one volume of Lysis Buffer, take it off the ice, and solubilize the urea with the palm of the hand.
  • Do not overheat since this can result in modifications of the protein. Samples can now be used directly or stored at -80oC until needed.


    One-Diemensional Isoelectric Focusing Gel Electrophoresis

    6. Thoroughly clean glass tubes (1-1.5 mm internal diameter and 10-15 cm long; 1 ml glass pipette can be used) in chromic acid.

    7. Rinse glass tubes in following order.
  • Rinse in deionized H2O.
  • Rinse in fresh KOH-Ethanol Solution for 20 minutes.
  • Rinse thoroughly with deionized H2O.
  • Rinse in 100% ethanol.
  • Air dry.

    8. Seal the glass tube bottoms with three layers of parafilm.

    9. Line up glass tubes vertically around a 500 ml beaker with elastic bands.

    10. Mark the tubes to the same point with a felt-tipped pen to ensure that gel lengths are uniform.
  • (This is important to facilitate reproducibility between runs).

    11. Make isoelectric focusing gel solution as follows.

    Total Volume
    5 ml
    10 ml
    15 ml
    Urea (Final conc. = 9.0 M)
    2.75 g
    5.5 g
    8.25 g
    30% Acrylamide:Bis Solution for IEF (19:1)
    0.667 ml
    1.334 ml
    2 ml
    50% Igepal CA-630
    100 ul
    200 ul
    300 ul
    Ampholines (40%, pH 5-7)
    50 ul
    100 ul
    150 ul
    Ampholines (40%, pH 3-10)
    250 ul
    500 ul
    750 ul
    Deionized H2O
    2 ml
    4 ml
    6 ml

  • Final conc. of ampholine should be 2%.
  • Ampholine composition may be changed depending on targeting protein's pI.

    12. Dissolve the urea by swirling in a water bath whose temperature is set not higher than 37oC and then briefly degas under vacuum,

    13. Add ammonium persulfate and TEMED as follows.

    Total Volume
    5 ml
    10 ml
    15 ml
    10% Ammonium persulfate
    24 ul
    48 ul
    70 ul
    TEMED
    3.4 ul
    6.8 ul
    10 ul

  • Mix briefly and use immediately.


    14. Using a Pasteur pipette, fill the tubes to about 1 cm from the top, being careful to avoid trapping air bubbles.
  • (Air bubbles may be removed by carefully tapping the tubes).

    15. Overlay the gel mix with 10 ul of Sample Overlay Buffer, allow it to polymerize overnight (or, at least >3 hrs).
  • Overnight polymerization gives better resolution.

    16. Carefully remove parafilm to avoid damage to the bottom of the gel.

    17. Place the gels in the electrophoresis tank.

    18. Fill the lower chamber with extensively degassed Anode Electrode Solution.

    19. Remove any trapped air bubbles from the end of the gels by a gentle stream of fluid using a bent syringe.

    20. Remove the water from the top of the gel with a Pasteur pipette and replace with 10 ul Lysis Buffer, 10 ul Sample Overlay Buffer and 20 mM NaOH to fill the tubes.

    21. Add the extensively degassed Cathode Electrode Solution carefully to the upper chamber and connect to the cathode and the lower chamber to the anode.

    22. Pre-run the gel at 200 volts for 20 minutes, 300 volts for 30 minutes, and 400 volts for 1 hour.
  • Do not cool the gels during this procedure since the urea will crystallize out.

    23. Remove the Cathode Electrode Solution from the upper chamber.

    24. Remove the liquid from above the gels and wash the tops with three washes of 20 ul of water.

    25. The samples are loaded in a volume of 5-50 ul with a syringe and overlaid with 10 ul Sample Overlay Buffer and 20 mM NaOH to fill the tubes.

    26. Fill the upper electrophoresis chamber with the Cathode Electrode Solution.

    27. Electrophorese for 3-18 hours at 400 volts.

    28. One hour before the termination of the run, turn the voltage up to 800-1,000 volts to increase the band sharpness.
  • But do not exceed 10,000 volts-hour since bands will become distorted.

    29. Turn off the power-pack and wait a few seconds, then remove the tubes and force the gels out onto parafilm troughs with a syringe full of water connected to the tubes via a flexible plastic tubing.

    30. Put the gels into capped tubes containing 5 ml of SDS Sample Buffer. Leave for minimally 20 minutes without agitation and at this point the gels may be stored at -70oC indefinitely.
  • After defrosting, it is advisable to exchange the SDS Sample Buffer and leave for a further 30 minutes before loading onto the second dimension.


    Two-Dimensional SDS-Slab Gel Electrophoresis

    31. Assemble the slab gel apparatus. Make it level and vertical.
  • Wash the plates thoroughly as follows.
  • Wash in 5% Decon.
  • Rinse with deionized H2O.
  • Rinse with ethanol.
  • Air dry the glass plates.
  • Do not wipe with tissues that leave lint since this will interfere with gel polymerization.


    32. The SDS Separating Gel is made as follows for 40 ml, 10-20% gradient gel.


    10% SDS Gel
    20% SDS Gel
    40% Acrylamide:bis Solution for SDS-PAGE (37.5:1)
    10 ml
    20 ml
    10% SDS
    0.4 ml
    0.4 ml
    Deionized H2O
    19.6 ml
    9.6ml
    4x Separating Gel Buffer
    10 ml
    10 ml
    Total volume
    40 ml
    40 ml
    TEMED
    70 ul
    70 ul
    10% APS
    300 ul
    300 ul



    33. Mix and use
    immediately.
    Pour the gel down the side of the gel plates to about 2.5 cm from the top.

    34. Overlay with H2O-saturated butanol applied at one end.

    35. Polymerize for 1 hour until the gel interface can be seen as a sharp straight boundary.

    36. Prepare the Stacking Gel Solution as follows.

    40% Acrylamide:Bis Solution for SDS-PAGE (37.5:1)
    1.2 ml
    Deionized H2O
    6.2 ml
    10% SDS
    0.1 ml
    4x Stacking Gel Buffer
    2.5 ml
    Total volume
    10 ml
    TEMED
    20 ul
    10% APS
    70 ul


    39. Mix and use
    immediately.
  • Wash the gel surface with water, and pour the Stacking Gel Solution from Step 38 up to a level 1 mm below the gel plate edge before inserting a Teflon edge.

    40. Overlay with water-saturated butanol and allow this solution to polymerize for 1 hour.

    41. Remove the Teflon strip, rinse the surface of the gel with water.

    42. Load the first dimension IEF gel on top of the second dimension SDS gel.
  • Take the defrosted, re-equilibrated IEF gel and straighten it in a trough of parafilm. Remove the liquid and apply it directly to the top of the stacking gel.

    43. Press down gently with a curved spatula tip from one end to ensure the removal of air bubbles, but be careful not to stretch the gel since this is one stage where variability can be introduced into the procedure.

    44. Overlay the gel with 1 ml 2 % agarose in SDS Sample Buffer.

    45. Allow it to set for 5 minutes.

    46. Assemble the slab gel system, fill the reservoirs with 1 x Tris-Glycine-SDS Running Buffer, and remove any air bubbles trapped under the gel with a bent syringe needle.

    47. Start the electrophoresis at 20 mA/gel for 8-9 hours until the bromophenol blue reaches the bottom of the gel.
    It is essential to avoid overheating and thus if the gels are run fast, the whole lot can be run in the cold room.
  • For Bio-Rad Protean II xi system (20 cm x 16 cm x 1.5 mm gel), total volt.hour = 1,200 - 1,300 V.H

    48. Optional: After 1-2 hour, change the upper reservoir buffer (or alternatively, continuously mix the upper and lower reservoirs with a pump-siphon system) to prevent vertical tailing of the protein spots.

    49. At the end of the run, turn off the power, remove the plates, separate them with gentle leverage.

    50. Fix the gel in 100 ml of Gel Fixer for 1 hour.

    51. Stain the gel with 100 ml of Gel Staining Solution for 1 hour.

    52. Destain the gel using several changes in the Gel Fixer.

    53. Dry the gel using a gel drier and autoradiographed for several different lengths of time.
  • For UV crosslinking, expose for 1 week.




    NOTES

  • Use the highest quality urea and prepare it fresh. Proteins may be carbamylated by isocyanate impurities in the urea solution. Prerun the isoelectric focusing gels to remove isocyanate contamination. It is also advisable to have ampholines present wherever proteins are in contact with urea. Basic ampholines may precipitate nucleic acids, with the resultant binding of protein producing streaking, but this may be easily solved by treating the sample with ribonuclease and deoxyribonuclease.

  • Vertical streaking can also occur and this is caused either by not changing or recycling the running buffer or, more commonly, by poor equilibration of the first-dimension gels. Occasionally, horizontal streaking can occur owing to poor solubility of proteins, but high concentrations of NP-40 and urea usually increase solubility sufficiently.

  • Spot size increases nonlinearly with high protein concentrations and overloading may cause precipitation at the top of the isoetectic focusing gel, which can streak across the pH gradient. Overloading may also cause pH inversions in the isoelectric focusing dimension. If samples are too dilute or at too low a specific activity, lyophilize them followed by solubilization of the pellet directly in lysis buffer. But remember that any salt will also be concentrated by these techniques and the salt may cause artifacts on the gel.

  • The trypsin used in cell culture may carry over and cause streaking and artifactual gel patterns.

  • Ampholines also run as small proteins, are acid precipitable, and will stain. They can be eluted from the gel using the fixative described above and so avoid masking the detection of small proteins migrating near the gel front.

  • Poor polymerization of gel is usually caused by dirty plates or poor quality bisacrylamide or acrylamide.

  • SDS slab-gel electrophoresis separates according to molecular weight since SDS binds to most proteins on a molar basis (1.4 to 1) giving a uniform charge-to-mass ratio. Thus, dependent on the size range of the proteins to be analyzed, a suitable percentage acrylamide gel may be selected.


    Solubilization of proteins forf IEF
    This is achieved by the use of chaotropic agents (such as urea), detergents (such as CHAPS), reducing agents (such as dithiothreitol), buffers and ampholytes (the charged species responsible for the pH gradient in isoelectric focusing). There are only a small number of chemicals which can satisfy these criteria, along with other important requirements such as the minimising of ionic strength (a low ionic strength allows high voltages to be applied during isoelectric focusing. This allows for a faster, more efficient focusing step). Comprehensive details of solubilization methods can be found elsewhere [9] [11].

  • Chaotropic agents
    Chaotropic agents disrupt hydrogen bonds. This disruption occurs both in the proteins and the water used to make the solubilizing buffer. Chaotropic agents prevent unwanted aggregation and formation of secondary structure which can alter protein mobility.
  • Detergents
    These disrupt hydrophobic interactions between species, as well as promoting solubility. Detergents must be non-ionic or zwitterionic to prevent them migrating during the isoelectric focusing step.
  • Reducing agents
    These disrupt disulfide bonds between cysteine residues. This allows analysis of single subunits of protein.
  • Carrier Ampholytes
    Some proteins tend to precipitate at their isoelectric point. To prevent this one may add salt to the protein solution. However as soon as one performs isoelectric focusing the salt will migrate away from protein promoting precipitation. To solve this problem carrier ampholytes are often included in the buffer.
  • Removal of nucleic acid
    The presence of nucleic acids, especially DNA, interferes with isoelectric focusing of proteins. Under denaturing conditions DNA complexes are dissociated and markedly increase the viscosity of the solution. This inhibits entry of the protein to the gel matrix and slows their movement during focusing. Nucleic acids can be removed by enzymatic digestion.
  • Keratin
    Careful handling is important to mimimise contamination from hair and skin.

    KIT INFORMATION




    REFERENCES

  • Celis, J., and Bravo, R. (eds), 1983, Two dimensional gel electrophoresis of proteins. Academic Press, San Diego, USA.

  • Duncan, R., and McConkey, E.H., 1982, How many proteins are there in a typical mammalian cell? Clin. Chem. 28, 749-755.

  • Kalischmidt, E., and Wittmann, H.G., 1970, Ribosomal proteins. VII. Two dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Ann. Biochem. 36, 401-412.

  • O'Farrell, P.H., 1975, High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021.

  • O'Farrell, P.Z., Goodman, H.M., and O'Farrell, P.H., 1977, High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142.

  • Pollard, J.W., 1983, Application of two-dimensional polyacrylamide gel electrophoresis to studies of mistranslation in animal and bacterial cells in Two-Dimensional Gel Electrophoresis of Proteins (eds Celis, J., and Bravo, R.), Academic Press, pp. 363-395. San Diego, USA.


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