Molecular Weight Standards
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I. BACKGROUND & PURPOSE
Gel electrophoresis is a useful method to separate and/or identify proteins and nucleic acids. In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins are separated largely on the basis of polypeptide length, and so their molecular weight can also be estimated. SDS does however denature the protein, so activity stains cannot be used to identify particular enzymes. Described below is the protocol for preparing and using Laemmli discontinuous gels. In this system, two sequential gels are actually used; the top gel, called the stacking gel, is slightly acidic (pH 6.8) and has a low (5.5%) acrylamide concentration to make a porous gel. Under these conditions proteins separate poorly but form thin, sharply defined bands. The lower gel, called the separating, or resolving gel, is more basic (pH 8.8), and has a higher polyacrylamide content (in our case, 12%), which causes the gel to have narrower channels or pores. As a protein, concentrated into sharp bands by the stacking gel, travels through the separating gel, the narrower pores have a sieving effect, allowing smaller proteins to travel more easily, and hence rapidly, than larger proteins.
II. SAMPLE PREPARATION
For best results, all samples should be in identical, low ionic strength
1. Mix 50 µL of each sample with an equal volume of one of the denaturing buffers below.
2. Heat in a boiling water bath for one minute. In most cases, brief boiling (1 -2 min.) improves denaturation, but it may also cause the protein to precipitate.
Denaturing Buffers (DB)
|Components||DB I .||DB II|
|Tris.HCl||0.25 M||0.0125 M|
|SDS||2% (w/v)||2% (w/v)|
|b-Mercaptoethanol||2% (v/v)||5% (v/v)|
|Bromophenol blue||0.001% (w/v)||0.001% (w/v)|
1. Remove the comb and clamp the gel to the electrophoretic apparatus.
2. Fill the top electrolyte compartment with running buffer.
3. Check for leaks from the top into the bottom compartment. If there are no leaks, fill the bottom compartment.
4. With a plastic Pasteur pipette, thoroughly rinse each well in the stacking gel with running buffer.
5. Apply the sample by using a micropipette to carefully add up to ~25 µL of protein in DB1 or DB2 to the bottom of a well. The volume and protein concentration of the sample should be sufficient to give at least 10 µg of each protein.* If possible, avoid using the end wells.
6. Apply 15 µL of the molecular weight standards to one or two wells, preferably in an asymmetric position, to allow the front and back of the gel to be identified later.
7. Carefully record the contents of each well.
8. Replace the cover of the electrophoretic cell, with the (+) symbol on the cover connected to the (+) on the cell, so that the anode (+) is the bottom electrode.
9. Check the electrical connections on the cell to ensure that solution is not in contact with either banana plug, and connect the anode to the (+) terminal on the power supply, and the cathode to the negative terminal. (Notice the convention inversion for electrodes: + is the anode, and - the cathode).
10. Apply 15 mA/gel until the proteins are well into the stacking gel, then 35 mA/gel until the tracking dye reaches the bottom of the gel (about 45 minutes in this system).
11. Always turn down the power and unplug the wires from the power supply before removing the cover.
* It is often useful to apply different sample volumes to several wells, so at least one lane has bands that are detectable but not overloaded.
|Tris base||0.25 M||36.3|
|Dilute 10-fold before use. Replace if the final pH is not within 0.1 pH units of pH 8.3.|
IV. TOTAL PROTEIN STAINING OF ELECTROPHORETIC GELS
Gel staining can modify the electrophoretic properties of the proteins and so may interfere with protein transfer during Western blotting. For this reason, it generally is not advisable to stain a gel that is to be used for a Western blot. It is useful, however, to stain the gel after performing a Western blot, to ensure that the protein has successfully transferred from the gel to the membrane. Once you've performed the Western blot, follow the protocol below to do the total protein stainning of your gel.
Protein Staining Solutions