Cleveland Peptide Maps
References:

Laemmli,U.K. (1974) Nature 227, 680-685

Cleveland, D.W., et al. (1977) J. Biol. Chem. 252, 1102-1106

I. Solutions & Equipment:

Slab gel electrophoresis apparatus, 0.75 mm spacers
Laemmli discontinuous gel buffers, solutions

Gel Slice Buffer I:	Gel Slice Buffer II:
0.125 M Tris pH 6.8	0.125 M Tris pH 6.8
10 % glycerol	10% glycerol
	0.1% SDS
	1% 2-mercaptoethanol

Proteases:

Staph. V8 protease	dilutions to 1 ug/ml
Chymotrypsin	dilutions to 10 ug/ml
Trypsin	dilutions to 10 ug/ml
Papain	dilutions to 10 ug/ml

All protease dilutions should be made in Gel Slice Buffer I with added Bromphenol Blue so that the solutions are blue.

II. Preparative Gel Electrophoresis:

1. Prepare a standard Laemmli gel of 0.75 mm thickness or a microgel of 0.5 mm thickness.

2. If you have lots of sample, pour a continuous well instead of several wells in the stacking gel.

3. Load the sample at a concentration known to resolve the proteins in the mixture and run the gel as usual.

4. Stain the gel for 5 minutes in Coomassie Brilliant Blue or until you can see the bands. You do not want to fix the proteins, so the less time in this solution the better.

5. Destain in 7.5% methanol/10% acetic acid briefly, 2 or 3 washes, 5-10 minutes total time.

6. Place gel on new saran wrap on top of a light box. Wearing clean gloves, and using specially cleaned utensils, excise the band with a new razor blade, lift up and place in a new 15 ml plasticware screw cap tube. Add 10 ml of Gel Slice Buffer I or Gel Slice Buffer II and shake for 30 minutes.

7. Pour off or aspirate off most of the buffer. Place in the -20o C freezer until the map is to be made.

8. Prepare a standard Laemmli gel which will provide separation fragments you anticipate that proteolysis should generate. Wells should be about 5 mm wide and 15 mm high (tall & skinny).

9. Design your peptide map. Run side by side the proteins which you are comparing in groups: first, no protease; second, low protease concentration, third, higher protease concentration, etc. Protease concentrations depend upon the activity of the protease & susceptibility of the protein to cleavage. As a general rule, 1-50 ng of Staph.a. V8 is a good range, while 10-100 ng of other proteases are a good range to try per well. Always run a lane with the protease by itself @ the highest concentration used so that you know which bands are from the protease.

10. With the gel upright before placing the gel in the apparatus, mark the wells and remove the comb. Remove gel slices from the freezer, and when thawed, pour onto a new piece of parafilm. Cut segments slightly narrower than the wells. Depending upon the protein concentration, place 1-3 slices in each well.

11. Place 5-10 uL of 2X sample buffer in each well, enough to cover each slice. Carefully fill each well with running buffer to the top. Place gel in electrophoresis apparatus and fill chambers with running buffer.

12. Add protease to the wells. It should layer on top of the 2X sample buffer, but not touch the slices.

13. Start electrophoresis as usual. When the dye front reaches the interface between the stacking & separating gels, shut off the current for 30 minutes. Resume run as usual. Silver stain gel.

14. One common problem with this procedure is proteolysis of the gel slice during handling. All care must be taken when preparing solutions and handling the slices to avoid contamination by proteases. Preparing picture perfect peptide maps is a combination of art & luck. If you have a pretty map the first time, take advantage of the photo opportunity! Good luck.

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