Tissue prepatation for WB and other issues - (Jun/06/2005 )

Hi I'm new bird in WB, and I have some troubles running WB technique.

So my first question is: what is the best way for the tissue preps. What I'm doing is homogenizing fresh tissue in camiolo buffer with 10ul/ml protease arrest solution?
Then spin and remove particulates and quantitate with Bradford assay..

I have bands in my +ve as well as my -ve controls, and faint or no bands in my unknowns or nonspecific bands...

Thank you.

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another question,

do I have to add b-meEtOH (fresh) to the loading buffer every time I'm running my samples?? because what I did was adding 0.2% of the reducing agent to the loading buffer stock that I have??

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I really need your help guys,

Ok here is another problem, I always have apparently specific bands of unanticipated molecular weight and they are of lower molecular weight than my target??? I’m searching the net and found that this could be attributed to Proteolytic degradation??? Is that possible and if so how can I overcome this problem…

I truly believe that the source of all the problems I’m facing in my WB is from the first step which is the tissue preparation…

Please help me….

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Hi all,

Do you think that Sonicating the protein might help improving the signal...

Please..help..

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Hi,

I typically grind the tissue in liquid N2, collect the grinded tissue sample, then add lysis buffer (camiolo buffer in your case).

Reducing agent should be added freshly to the SDS sample buffer. If you add the reducing agent into the SDS sample buffer stock, I'd freeze them in -20 deg. C.

I do not know how big your tissue is, I do not know how stable your protein of interest is, I do not know how you are homogenizing, and if proteolytic degradation is the case, then perhaps protease inhibitor is not penetrating the cells fast enough.

Good luck.

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Hi vagrants,

Thank you so much for answering my question, and with regard to the tissue am testing they are 50-100mg from mice neonates.

I collect the tissue directly in camiolo buffer after freshly adding the protease arrest to the buffer and homogenize in ice for about 2min. and then centrifuge a @ 3000 rpm for 15 min.

I hope I gave you enough information, and if you can direct me more in this issue.

And another issue what is the typical diluents you usually use for diluting the protein lysate, like e.g. I want to load 25ug/well and that is 6ul of the protein then add 4ul of ??? to make 10ul and add 10ul of loading buffer

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