I checked the degree of proteases actions on protein samples by performed SDS-PAGE from muscle tissue sample,i loaded the sample on 4 different incubation time at RT without using any inhibitors!!
I noticed that no difference between the bands in all time interval point(the bands in 5 minute incubation look the same of 4hr incubation,is that make sense?Or is the action of proteases are very minimal and slow?!!
Eric, it is not possible to address your questions without information about your proteases, e.g. what is their pH optimum in terms of structural and enzymatic stability, how senstive are they to detergent and reducing agens?
If I understand it correctly Eric, you are interested in knowing the effect of endogenous proteases over time. In this regard it would be useful for us to know the components of homogenization buffer, as Erwin mentioned above detergetnts, denaturing and reducing agents affect enzymatic activity.
Thanks Erwin and Paschalis,i prepared laemmli sample buffer 65 mM Tris HCL;PH 7.8, 10%glycerol, 2%SDS, 0.025% bromophenol.i took 950 ul of this buffer with 50 ul mercaptoethanol.
I have to dilute my sample1 :1 with this buffer and reducing agent but actually i did not know well the volume of my sample because its collected in frozen so the ratio is not accurate 1:1 100%
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