V8 Peptide Mapping (Cleveland-Laemmli digest)
This technique serves to show identity, or at least close similarity, between two proteins
represented by bands on an SDS-PAGE gel.
The original protocol was published in “Cleveland, D., Fischer, S., Kirschner, M., and
Laemmli, U.: Peptide Mapping by Limited Proteolysis in Sodium Dodecyl Sulfate and
Analysis by Gel Electrophoresis, J. Biol. Chem., 252, 1102 (1977)”
1. Carry out in vitro kinase reaction as usual and resolve proteins on SDS-PAGE. Dry
down the gel and expose to film for the minimum amount of time that allows the
bands to be clearly visualized. Be sure to have a marker so that you can unambigously
line up the film with the dried down gel and identify lanes and locations of bands.
Tape the film to the gel on all 4 sides so it doesn’t move around while cutting.
2. Pour the second dimension SDS-PAGE gel (usually 12-15 %, make a longer stacking
gel – 3 cm from the bottom of the wells to the separating gel, include 1 mM EDTA in
all gel mixes !)
3. Prepare the following buffers: Overlay / Rehydration buffer: [125 mM Tris pH 6.8, 1
mM EDTA, 0.1 % SDS, 30 % glycerol, 0.0075 % Bromophenol blue] Add 2.5 mM
DTT right before use !
Enzyme buffer: [125 mM Tris pH 6.8, 1 mM
EDTA, 0.1 % SDS, 10 % glycerol, 0.0075 % Bromophenol blue] Add 2.5 mM DTT
right before use !
4. Take out Staphylococcus aureus V8 protease from the –80 ˚C freezer (it’s a 500
µg/ml =500 ng/ µl stock in [125 mM Tris pH 6.8, 1 mM EDTA]) short time in
advance and thaw on ice.
5. Excise the band of interest (one by one …) using a scalpel with a sharp blade and
rehydrate the gel piece 1-2 minutes in appr. 100 µl rehydration buffer (with DTT).
Try to cut the gel band through the film trying to avoiding getting the backing paper
as well. If this is not possible you may get rid of the backing paper upon rehydration
using forceps.
6. Carefully place the gel piece in the well of the second gel using forceps (running
buffer has been added to the wells). Align the gel piece carefully along the bottom of
the well using a long gel-loading tip. Overlay with 10 µl of rehydration/ overlay
buffer. Repeat for the next gel slice and so on.
7. Add for each well 10 µl of enzyme buffer containing the appropriate amount of V8.
Try dilutions in the range 5-500 ng (in a 10 µl volume). In our case, 10 ng and 50 ng
seems to be appropriate. Make up the V8 enzyme dilution right before use as
follows: 10 ng amoun t: 2 µl V8 stock into 1000 µl enzyme buffer (with DTT !); 50
ng amoun t: 5 µl V8 stock into 500 µl enzyme buffer (with DTT). Don’t refreeze the
V8 aliquot.
8. Run the gel at 125 V two-thirds of the way through the stacking gel, then turn off the
power to allow V8 digestion for 30 min; finally resume running the gel overnight at
50 V.
9. Dry down the gel and expose to film using a screen and storage at –80 ˚C during
exposure.