Protein Protocols Protein Protocols

Immunoprecipitation 1099
Table 2
Protease Inhibitors Often Used in Lysis Buffers
Starting
Inhibitor Specificity Solubility/stability concentrations
Antipain- Trypsin, papain, Soluble in H 2O, 50 µg/mL
dihydrochloride plasmin methanol DMSO.
Dilute solutions
stable 1 mo at –20°C
Aprotinin Plasmin, trypsin, Soluble in H 2O, 0.06–2 µg/mL
chymotrypsin aqueous buffers.
Dilute solutions
stable 6 mo at –20°C
Chymostatin α, β, γ, δ-Chymo- Soluble in glacial 0.1 ng/mL
trypsin acetic acid, DMSO.
Dilute solutions
stable 1 mo at –20°C
E-64 Papain, Soluble in a 1:1 0.5-10 µg/mL
cathepsin B, L mixture (v/v) of
ethanol and water
Leupeptin Trypsin, Soluble in H 2O. 0.5 µg/mL
papain, plasmin, Dilute solutions
cathepsin B stable 6 mo
at –20°C
PMSF Chymotrypsin, Soluble in 2-propanol 17–170 µg/mL
trypsin, Stock solutions >100 mM (0.1–1 mM)
thrombin, PMSF in 100% 2-propanol
papain stable at room
temperature >1 yr
proteins and secreted proteins generally being more resistant than cytoplasmic proteins.
Therefore, it is advised to work on ice and include a protease inhibitor in the lysis
buffer. Proteases are divided into five classes according to their mechanism of catalysis:
serine proteases, cysteine proteases, aspartic proteases, metalloproteases, as well
as enzymes with unknown reaction sites (4). Among the two most commonly used
protease inhibitors, aprotinin is a serine protease inhibitor and phenylmethylsulfonyl
fluoride (PMSF) is a serine and cysteine protease inhibitor (5).
Unlike other compartments in the cell where protein folding occurs, the endoplasmic
reticulum is oxidizing and therefore promotes the formation of disulfide bonds.
Although the cytosol of intact cells is reduced, and therefore prevents formation of
disulfide bonds, disruption of cells without previous treatment with alkylating agents
such as N-ethylmaleimide (NEM), will incorrectly introduce disulfide bonds in cytosolic
proteins (6). To avoid artificial disulfide bond formation during cell lysis, cells
should be washed with ice-cold phosphate-buffered saline (PBS) containing NEM and
lysed in lysis buffer containing NEM.