protein solubilization and quantitation after extraction using Trizol - (Mar/21/2012 )
I have isolated RNA, DNA and protein from the same tissue sample using the Trizol method.
I am having trouble getting the protein into solution. I followed the protocol and used 1%SDS and heated at 50C overnight, I then added lamelli buffer (without bromophenol blue) to help solubilize the samples. Some samples went into solution while others did not.
I need to run these samples on a Western blot. The samples are various rodent tissue. I was told that I cannot use Bradford assay to quantitate as there is SDS and DTT present. I have access to the DC protein assay. Will that help?
Is it necessary to quantitate samples before running on western? Can I just do a few dilutions of each sample?
Also does anyone know the expected yield of protein per mg of tissue homogenized? I cant seem to find it anywhere on the web.
I have read that people dont get good results using Trizol method for protein extraction. What other methods would people recommend?
I have not seen anyone use Trizol for protein extraction although it could be possible.
There are several popular lysis buffers for tissue. My suggestion is that you start with a buffer called RIPA with your homogenized tissue. You can find its recipe online. or search my previous posts. However RIPA is a strong lysis buffer. There are some milders ones too. I normally use CHAPS buffer myself for my cultured cells. I also remember a friend of mine used Lysis Buffers (M Buffer?) from Pierce (now Thermo) for homogenized tissue.
to answer your first question, yes, it is better to know the concentration of your sample. With Biorad's Mini Protean3 we load between 10-60 ug in each well.
if you have a microplate reader in your lab you can easily run Bradford assay. It is really easy and I recommend it for relative quantification, and I honestly don't know why people go for expensive commercial kits. you can find my protocol in my previous posts.
Also, you can use RIPA or many other lyssis buffers with Bradford's method. Addition of protease inhibitor to your lysis buffer also won't affect your reading much.
you can use the thermo-pierce protein assay selection guide to decide which may be the best protocol for your purpose.