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It is possible to see three bands for the large subunit of cleaved caspase-3. The bands all represent cleavage at Asp175. However, the distinction between the three is the extent of processing at the amino termini of the proteins [see Fernandes-Alnemri, T. et al. (1996) Proc Natl Acad Sci U S A. 93(15), 7464-9 (PMID: 8755496; http://www.ncbi.nlm.nih.gov/pubmed/8755496)]. Processing of caspase-3 into its fully active form is a two-step process. Step 1 involves cleavage at Asp175, which generates the 20 kDa large subunit and the 12 kDa small subunit. In step 2, the 20 kDa large subunit is further cleaved at Asp9, probably through auto-catalytic activity. The removal of the first nine amino acids of the pro-domain results in the 19 kDa subunit. Further cleavage can also occur more slowly at Asp28 to generate the fully mature 17 kDa subunit. Therefore, any accumulation of the 20 and 19 kDa subunits is presumabl…
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CIP/Lambda Phosphatase Treatment of Cell Lysates:
1) Wash cells 2X with ice-cold PBS.
2) Add cold 1X cell lysis buffer that does not include phosphatase inhibitors [1-2 E7 cells per ml or 0.4ml per 10 cm plate (80-90% confluent)]. Our cell lysis buffer is prepared as follows:
20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton
1 ug/ml leupeptin
1mM PMSF
3) Add 20ul Quick CIP (NEB #M0525; https://www.neb.com/products/m0525-quick-cip#Product%20Information) per 400ul of cell extract.
4) Incubate with Quick CIP at 37C for 1 hour.
5) Add 20ul lambda protein phosphatase (CST