I have used RIPA buffer following the protocol listed on a Nature paper. But I have not been able to detect the protein of interest. Have tried three different sources for the antibody and also included a knockout lysate but the data is still messy and inconclusive. I wanted to know what is the best way to be able to detect membrane proteins by western blot. Should I try membrane fractionation kit or milder lysis buffer like NP40 or something else. Any help would be appreciated. Thanks in advance
When you prepare the samples for SDS-PAGE, try skipping the heating step. Some membrane proteins are lost when the SDS-PAGE sample is heated supposedly.
Hello Vineeth Kumar Murali
Membrane proteins are insoluble in aqueous media as a result of incompatibility between the hydrophilic property of the solvent molecules and the hydrophobic nature of membrane protein surfaces. Depending upon the hydrophobic property of your membrane protein of interest, you may use different concentration of detergents. Non-ionic and zwitterionic detergents are milder and less denaturing than ionic detergents and are used to solubilize membrane proteins.
You may refer to the article attached below for the protocol to prepare cell lysate to detect membrane proteins. It may be helpful.
https://www.jstage.jst.go.jp/article/bbb/71/9/71_70342/_article
Further, hydrophobic proteins are sensitive to temperature. So, avoid boiling them at temperatures >90-95 degree C as they will aggregate at high temperatures. The samples may be heated at 45-50 degree C, or you may incubate the samples for 1h at 37 degree C.
Best.
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