Determination of protein concentration is very important befor loading the samples in wells. So, can you please tell me the most effective and easy method by which protein can be quantified in tissue lysate.
Is there any interference of proteases inhibitors or lysis buffer contents on the determination of protein content by any method. As earlier i experienced the problem in determination of protein content in tissue lysate in lysis buffer + protease inhibitors by Lowery method.
If can you give me the full method of determination of protein content starting from tissue collection then its lysis and then determination.
Hi Vinary I think Bradford or BCA protein assay will be helpful either manually or using kits as in attachment files
Thanks.. but i want to know that is there any interference of the lysis buffer and protease inhibitors in the estimation of protein conc in tissue sample
Dear Vinay, I use BCA kit from thermo scientific for measuring protein conc in cell lysates. It's easy to use with Nanodrop and for me, it works well with RIPA buffer (with detergents). However, if it seems to interfere with your lysis buffer, you can dilute your samples for protein concentration determination and multiply with the dilution factor afterwards. I hope it helps
I also use the BCA thermo scientific kit and Nanodrop, I dilute my samples 1:10 (T-PER lysis buffer for tissues) and then multiply with the dilution factor afterwards.
Dear Jana, what is T-PER lysis buffer. Can you tell in detail in step wise way. How you proceed for protein determination after taking tissue
It is a commercial lysis buffer for tissue samples sold by Thermo Scientific, I just follow their protocol (see link). The second link shows the protocol for the protein determination - I use the high range assay.
http://www.piercenet.com/product/t-per-tissue-protein-extraction-reagent
http://www.nanodrop.com/Library/T085-NanoDrop-2000-2000c-BCA%20Protocol.pdf
Dear Samra, do you add some additional protease inhibitors along RIPA buffer for lysis of tissue.
Can you tell me the ratio or proportion of tissue sample and RIPA buffer. This will help me. My tissue sample is skin/granulation tissue.
Our lab uses the microBCA or BCA Assay to determine protein concentration.
Dear Vinay, I use protease and phophatase inhibitor tablets from Roche. I can send you the product codes, if you want. The volume of RIPA (or any other tissue) lysis buffer depends on the weight of tissue. I usually use 300ul for half of mouse spleen
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