First ensure the separation of your protein in the SDS Gel with suitable protein marker. You will know that your protein is intact or deformation due to your purificatiom process/ heating ect..

Ensure the correct placement of gel and membrane in the transblot apparatus.

BSA will be the best blocking agent.

Ensure your antibody binds with your protein by simple precipitation/diffusion method. And check the manufacturer catalogue that the antibody has the FC portion.

Ensure the correct selection of secondary antibody conjugate ( check developed host system and your antibody & host system).

Ensure the selection of Substrate system corresponding to your conjugate.

eg : alkaline phosphatase with BCIP/NBT.

Hello Lokesh Mishra

Membrane protein extraction is a challenging task. You need to consider the below factors while isolating membrane proteins from tissues.

1. RIPA buffer may not be convenient enough to extract membrane proteins and membrane-associated proteins. Extraction of membrane proteins from the lipid layer requires a fine balance and articulation through the properties of the chosen surfactants and the surrounding lipid molecules of the protein. Membrane proteins are insoluble in aqueous media because 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. So, a wide range of detergents and detergent concentrations may be tested for optimal effect.

2. Not just detergent concentration and type, many other factors, including the buffer concentration and temperature of the buffer used for making tissue or cell homogenate, can affect membrane protein extraction. Ionic concentration of the buffer is one of the decisive factors for solubility of membrane proteins.

3. Hydrophobic proteins are sensitive to temperature. So, avoid boiling them at temperatures >90-95 degree C as they will aggregate at high temperatures. You may incubate the samples for 1h at 37-degree C.

4. Membrane proteins are more susceptible to protease degradation. Therefore, they would require extra attention in stabilization (add protease inhibitors fresh in the lysis buffer just before carrying out the lysis process).

5. Sometimes, the protein of interest may be expressed on cell membrane at different time points. So, you need to obtain more information about the protein of interest.

Hope this helps!

Best.

Validate your experiment efficacy step by step. You may use a dot blot to confirm that you extracted your protein successfully and that antibody selection is affirmative along with the membrane of choice.

Secondly, before conducting the whole western protocol steps, I strongly suggest performing a transfer efficacy evaluation initially. Your target is highly hydrophobic and moving it around through reagents would be harder than any soluble proteins. After a successful SDS-PAGE, you may apply ponceau S staining to your membrane to see whether your target is transferred to blot or not at the correct MW shift. Other conditions such as probing, washing, and blocking come later when choosing your next troubleshooting.

Good luck

a few ways you can try to get your result: 1) the most of proteins from tissue are matrix and cytoplasmic proteins, you have to remove these proteins to enrich your membrane proteins. To do this, use H2O plus protein inhibitor to homogenize your sample, spin down to remove the soluble proteins and keep the pellet (membrane and nuclear proteins), add RIPA to dissolve pellet (it is better you do homogenization again). 2) try buy solution such as Bio-rad casein for wetern blot to increase the sensitivity of detection.