You can dissolve the pellet in SDS-PAGE sample buffer, since you are going to run it on a gel anyway.

Hi Griffin A. Beach

I agree with Adam B Shapiro . In addition, you can also try dissolving it using 50 mM or 100mM TEABC buffer.

Thank you

Varsha

Hi Griffin A. Beach Ammonium sulfate is not very efficient for integral membrane protein precipitation unless the sample has been stripped of lipids.

You could try TX-114 phase separation of the homogenized sample. That should enrich for integral membrane proteins.

https://scholar.google.com/scholar?hl=en&as_sdt=0%2C23&q=tx-114+phase+separation&btnG=

Steingrimur Stefansson I have had some concerns with lipids interfering with the migration of proteins. I am new to protein extraction and westerns, but have noticed substances that float after centrifuging and have read that this is likely lipids.

Despite the presence of lipids I have managed to get clear bands stained with Coomassie blue; however, I have so far only ran the supernatant with electrophoresis.

Thank you for the link, I will look into TX-114 phase separation!

Adam B Shapiro & Varshasnata Mohanty I am new to protein extractions so sorry for my naivety, but would SDS-PAGE sample buffer or TEABC buffer dissolve the crude pellet retrieved from centrifuging the initial crude extract after homogenizing tissue in a diluted acetic acid solution?

I only ask for clarity sake, as I have been thus far precipitating protein from the initial supernatant and then resuspending the pellet retrieved from the second centrifuge step in Tris buffer.

Thanks for the recommendations!

The only potential problem is that the acidity of the sample might be enough to change the pH of the sample buffer. The correct pH is important for the SDS-PAGE separation. If the blue dye in the sample buffer turns yellow, it means the pH has dropped.

I agree with Adam. On what you already have, if the SDS-PAGE sample buffer turns yellow, add 1ul at a time of 1M Tris base until it turns back blue.

Unless snail tissue is different somehow from other tissues, I would homogenize an amount of wet tissue weight with 10-15 volumes of 2% SDS in 50mM Tris using a sonication device or bead beater apparatus. This should give you a protein concentration over 1 mg/ml, and solubilze .most membrane proteins present. Heat to denature the proteins (10 min at 85C). You can measure the protein conc. in a SDS-compatible BCA protein assay, and then dilute the appropriate amount of sample with 1x SDS-PAGE Laemmli buffer for your Western.

Hi Griffin A. Beach

I agree with Mary Cristine Charlesworth. In my lab, for any tissue extraction, we use lysis buffer (4% SDS in 50mM TEABC) and use probe sonicator for the lysis. The tissue lysates were sonicated using probe sonicator followed by heating for 10-15 min at 90 o C. The lysates were then centrifuged and the cleared lysate was estimated using BCA assay.cIf you intent to run a SDS-PAGE, you can dissolve in 6x laemmli buffer.

In some cases, instead of using SDS lysis buffer, people use Urea or guanidine hydrochloride depending upon the tissue type. but in later cases, one has to take care of excess salts.

For precipitation, we often use ice-cold acetone and leave for 6-8 hours.

Varsha