could you comment on this Sample Preparation Protocol for SDS-PAGE from Muscle t - (Nov/20/2008 )
Sample Preparation Protocol for muscle tissue:
1. weigh around 0.1 gram of muscle sample powder to a 2 ml centrifuge tube. After weighing, add 10 times lysis buffer (based on the sample weight) and homogenize it immediately using a homogenizer for 5 to 10 sec. (question1: is 10 sec enough to lyse the muscle tissue?)
2. Place the samples on ice after homogenization, shorten this step as soon as possible.
3. Add equal volume of 2X sample loading buffer (37 mM tris-HCl pH 6.8, 4.4% SDS, 2.2% glycerol, and 0.002% Bromophenol Blue) and add 5% Mercaptoethanol to each sample (question2: Is it correct to add sample loading buffer in this step? My boss said 4.4% SDS can solubilize myofibril proteins during following boiling step. Those myofibril proteins are hard to extract during homogenization step)
4. Mixing well by vortexing and boil the samples for 5 min and then centrifuge for 5 min at 12,000 rpm.
5. Transfer the supernatant to another pre labeled centrifuge tube and store them at -80⁰C freezer for further testing.
Question3: I used to do like this: after homogenization for 2X10 seconds, incubate on ice for 20min, then do centrifugation and protein concentration measurement, then adjust volume according to protein concentration and add 1:1 2Xsample loading buffer, boil and run SDS-PAGE. My boss said incubate in lysis buffer on ice for 20min is totally wrong, I need add sample loading buffer quickly and boil it to avoid protease degradation. My protocol may not fit for muscle samples preparation, the above protocol is based on his experience. But I just doubt it because it is so different from the protocol I found on the Internet.
can someone give me a clue? Thanks!
Because My boss was unhappy about my method, I need evidence to support what I did makes sense. Or I need a third party to tell me his method makes sense. I appreciate your help!
your boss' protocol makes sense. muscle tissue is chock full of various proteases. so you want to inactivate them as soon as possible.
you can use your protocol if you have sufficient protease inhibitors in your lysis buffer.
you can use your protocol if you have sufficient protease inhibitors in your lysis buffer.
thank you! I can understand protease inhibition part. But am still confused why add 2Xsample loading buffer before centrifugation, does it really help to extract more protein from muscle during boiling?
the sample buffer (and boiling) will solubilize and denature the proteins in the homogenate. most of the proteases will be denatured, as well.