V8 PEPTIDE MAPPING (CLEVELANDLAEMMLI DIGEST) THIS TECHNIQUE SERVES TO

14 OF 14 AMINO ACIDS PEPTIDES AND PROTEINS AMINO
ADDITIONAL FILE 9 TETRAPEPTIDES PRESENT IN THE EXPANDED PLANT
ANTIBODY PRODUCTION RABBIT PEPTIDE COCKTAIL ORDER FORM AXIL SCIENTIFIC

BORIS ISOPEPTIDE TRANSCRIPT THE SIZE OF TRANSCRIPT (BP) MOLECULAR
CD14%20and%20a%20peptide%20thereof%20(pMO2)%20protect%20cells%20from%20apoptosis%20(provisional)
CELLPENETRATING PEPTIDE PENETRATIN ENABLES BRAIN DELIVERY OF THE PRPSCSPECIFIC

V8 Peptide Mapping

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.



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M INIREVIEW SMALL MOLECULE AND PEPTIDE INHIBITORS OF THE
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