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The following Cold Spring Harbor Laboratory Press protocols are brought to you by BioSupplyNet in a partnership with Sigma-Aldrich. A list of products is available to use in each protocol.
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Selected Protocol from "Protein-Protein Interactions", 2nd Edition, E. Golemis, P. Adam.
Direct Enzymatic Digestion of Protein Complexes
Sherry Niessen, Ian McLeod and John R. Yates III
Department of Cell Biology, The Scripps Research Institute, La Jolla, California
ABSTRACT
Protein complexes can be digested directly using a number of different protocols. The methods presented here include a double digest with endoproteinase Lys-C and trypsin, or a triple digest with elastase, subtilisin, and trypsin. Endoproteinase Lys-C cleaves carboxy-terminal to lysine residues, while trypsin cleaves carboxy-terminal to lysine and arginine residues. The triple digest uses the relatively nonspecific proteases elastase and subtilisin to create a large number of overlapping peptides for the analysis of posttranslational modifications (MacCoss et al. 2002b). The double and triple digest protocols begin with the reduction and alkylation of cysteine residues to disrupt disulfide bonds and, therefore, higher-order protein structure. |
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Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Acetone, cold |
65051 |
Acetone |
|
| CaCl2, 1 M |
21115 |
Calcium chloride solution |
|
| Formic Acid, 90% |
F0507 |
Formic acid |
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| Milli-Q water |
39253 |
Water, LC-MS CHROMASOLV® |
|
| Trichloroacetic acid (TCA), 20% |
T9159 |
Trichloroacetic acid |
|
| Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP), 100 mM |
C4706 |
Tris(2-carboxyethyl)phosphine hydrochloride |
|
| Iodoacetamide (IAA), 500 mM, prepare fresh |
I1149 |
Tris(2-carboxyethyl)phosphine hydrochloride |
|
| Tris-HCl (or Trizma base), 100 mM (pH 8.0) |
T6791 |
Trizma® base |
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| Urea, 8 M in 100 mM Tris-HCl (pH 8.0) |
U0631 |
Urea |
|
| Elastase Grade II |
E0127 |
Elastase, pancreatic from porcine pancreas |
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| Endoproteinase Lys-C |
P3428 |
Endoproteinase Lys-C from Lysobacter enzymogenes |
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| Subtilisin |
P8038 |
Proteinase, bacterial |
|
| Trypsin, sequencing grade, modified |
T8658 |
Trypsin from bovine pancreas, suitable for protein sequencing |
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The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
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