I tried to optimize conditions but havnt find any way. When i use mosquito cell line C6/36, target protein band comes at 50. But with live mosquito never got this. I attached membrane picture also please guide me how i can solve this issue Thanks.
You will have to add any standard control protein in this blot. Do you have one ?
As Mahesh already suggested, loading a control protein is always a good idea. You would easily see if your PA gels are running differently for any reason or if it has to do with the protein itself.
The different protein sizes could arise from your sample preparation. Maybe there is proteolytic cleavage in the mosquito preparation.
As you did not describe the sample preparation, I assume the protocols for cells and live mosquitos differ.
Have a look at your lysis protocols for the cells and the whole organism and compare the procedures. Make sure to include precautions against degradation in the live mosquito protocol. In general, always precool your solutions to 4°C, work on ice and add protease inhibitors or a inhibitor cocktail to all your buffers once the cells are disrupted.
Some chemicals like imidazole also show proteolytic activity, so check your buffer compositions for potential candidates.
For example, if your protein is tagged and the protocol includes an immobilized metal affinity chromatography as purification step (IMAC, usually Ni2+ or Co2+) with elution by imidazole-containing buffer, do not store your samples in the elution buffer. Remove imidazole by dialysis or ultrafiltration. From my experience, this is a crucial step and I would not store a protein in imidazole even if the samples are frozen at -80°C.
Finally, check if the samples used for PAGE have the same composition and are denatured in the same way. If the buffers of the cell and live mosquito preparation differ a lot, i.e. in ionic strength or maybe detergent content, this could lead to different electrophoresis patterns. I would then recommend to match the buffer composition by removing salts or detergents by dialysis or gel filtration.
I hope these suggestions give you starting points for troubleshooting!
Best, Marianne
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