I'm preparing to run Western blot analyses on rat tissue and have read some estimates where ~5mg of tissue is used with 300uL of lysis buffer. However, a hemisphere of a 4-month old rat weighs about 0.9g and therefore would require >30mL of buffer which is out of the question.
I have read about different lysis buffers besides RIPA that have been used and have an aliquot from a few fellow researchers (Tris-Triton buffer); again, I'm just not sure how much of this to use for my tissue in order to locate good amounts of my cytosolic proteins of interest (SOD-1 in particular).
I think the rule here is to have enough buffer to grinf your tissue and to resuspend what is extracted. I would try first with 5 to 10 mL and see what happens. Good luck
If I was planning on centrifuging the samples, wouldn't I need a consistent amount of tissue and lysis buffer? In our lab, we have done whole hemisphere mouse brains ( w/out the cerebellum) in 2mL of RIPA. However, with a new lysis buffer I guess my concern is whether I could just substitute the lysis buffer I'll use in place of the RIPA since RIPA isn't compatible with Bradford Assay.
Hi Stephen Lippi
Amounts of your buffer is equivalent with amounts of extracted protein.
when you increase buffer to tissue ratio, the concentration of dissolved target protein will be in steady state until a point. and after that comedown...
so
1- you can optimize your experiment( by measuring amounts of target protein in different ratio of buffer after extraction)
2- also you can use the amounts that have been tested by other researcher.
Article Effect of earthworm Eisenia fetida extract on blood and live...
You can homogenize your samples in 1:4 to 1:10 fold (sample weight : buffer volume), it will suit OK for wester blotting (WB).
If your targets are cytosolic proteins, you can homogenize in TRIS/EDTA-buffers or simillar ones, with protease inhibitors as these buffers will not prevent protease activity.
Maybe you could take an aliquot of the homogenate (and freezing at -80oC the remaining volume) and centrifuge it in microtubes to get the cytosolic fraction, because I believe that 1 g samples will get you large volumes of homogenates, what can be tricky to centrifuge at high speeds as you may need to use big tubes. A 1 ml aliquot would yield high amount of protein for WB.
After centrifugation, save an aliquot to run Bradford and continue with sample preparation for electrophoresis (adding glycerol, SDS, bromophenol blue...) to the sample, so the reagents will not interfere with your protein determination.
I hope that this may help you.
Cheers!
I usually use extraction/homogenization buffer (containing higher concentration of sucrose) at 1:9 ratio. After homogenization I usually save aliquots for BCA assay to know the protein concentration. The rest of the aliquots are stored at -80 C. Pls do not forget to add protease and phosphatase inhibitors in the buffer at the last moment. Hope it helps. Ranu
how much protein lysis buffer should be used for a mice retina and how should keep it in lysis buffer?
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