Solution Informationhelp
Enzyme: Complement C1s subcomponent
inhibitor: BDBM40932
substrate: n/a
 
Solution Type: Aqueous
pH at Preparation: n/a
Temp. Prep.: n/a
Comments: Materials Activated human complement factor C1s was purchased from Calbiochem (Cat #204879). Substrate Boc-Leu-Gly-Arg-AMC was from Bachem (Cat #I-1105.0050). Assay buffer consisted of 50 mM HEPES, pH 7.5, 0.2 M sodium chloride, and 0.2% polyethylene glycol (PEG). Low-volume 384-well black plates were from Corning (Item #3676). Assay Complement factor C1s (0.02 mg/mL) was incubated with Boc-Leu-Gly-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 2.5 hr at room temperature. IC50 was performed using a serial dilution of compounds from 50 uM to 75 nM. All IC50 determinations were done in triplicate. IC50 protocol 1.Serial dilute single compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM) 2.Fill low-volume plate with 4 uL water using Multidrop-micro 3.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384 4.Add 200 nL of compound (in DMSO from step 1) using Evolution pintool 5.Add 1 uL of Boc-Leu-Gly-Arg-AMC substrate (150 uM i
 
 
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