SDS-PAGE Western Blotting Protocols

Adapted from existing protocols by Vinh Pham
Last modified: June 5, 2003.
MATERIALS
All materials required are mentioned within the respective sections below.
PROCEDURE

Part I – Sample Preparation
1. Collect cells or fruiting bodies. Klett 100 cells are at a density of 5E8.
2. Pellet at RT for 1-2’ and remove s/n.
3. Extract protein by SDS/Urea Method, TCA-precipitation, or sonication.
4. Measure [protein] by Bradford (use 1-2 ul of each sample).

Part II – SDS-Polyacrylamide Gel Electrophoresis
1. For each SDS-PAGE gel, sandwich one small and one large glass plate,separated by spacers (smeared with silicone lubricant) and an alignment card.
Optional: The inside surface of the small glass plate can be treated with Rain-X to facilitate later removal of the gel.
2. Slide into holder without tightening the screws.
3. Place on pouring stand, making sure that the glass plates are pushed all the way to the bottom before tightening the screws. If the assembly is correct,then the whole set-up should snap into place when placed above the gray gasket.
4. Remove alignment card.
5. Slide in comb and mark a line at 2-3cm from the bottom of the comb. Make sure that the comb thickness is the same as the that of the spacers’.
6. Remove comb.
7. Pour resolving gel to the mark in step 5.

Optional: Create an agarose plug with newly liquified 1% agarose (i.e., the agarose needs to be hot when used). The agarose does not affect the running of the SDS-PAGE, although it does reduce the effective size of the resolving gel.

8. Add upper layer of water-saturated n-butanol to the top of the gel.
9. Let polymerize (this takes ~10-15’).
10. Invert gel to remove n-butanol. Touch with filter paper to wick off residual liquid.
11. Pour stacking gel to fill the remaining space between the glass plates.

12. Insert comb and let polymerize (this takes ~10-15’).
13. Prepare 20-30ml of sample, as follows:

14. Boil samples for at least 3’.
15. Place gel in holder/electrode, then transfer to running tank.
16. Fill with 1X Running Buffer (keep inside and outside buffer chambers separated).

Note: Mix by inversion in a Parafilm-covered graduated cylinder.Remove excess bubbles and foam with wads of paper towels.