Hi Sara Hawass

I think it might depend on how you prepared the cell lysate. I assume the GFP protein is easily isolated for the GFP only transfected cells because it is in the cytoplasmic fraction of the cell lysate. However, for ion channel-GFP transfected cells, I assume that you might be losing the ion channel-GFP because it is not liberated from the membrane (I think it is more challenging to isolate membrane-bound proteins).

Hi Mhd Yousuf Yassouf

Thank you very much for your answer. I think I agree with you because when i checked for the transfection the gfp only transfected cells were really bright green under the microscope, unlike the co transfected GFP with ion channel, cells were grean and all over the plate but pale grean.

But does this mean i lost my protein or i didn't get quite a lot of the ion channel protein? and what is your advice to prepare the protein lysate to isolate all proteins?

Thank you very much.

Best regards,

Are you assessing also a constitutive protein in your WB? the lack of protein band in the WB is limited to this specific sample ore is systemic?

What methos for protein extraction are you using?

Hi Sara Hawass

For the isolation of membrane-bound proteins (such as ion channels), you might need to use sonication or a stronger lysis buffer, adjust incubation time .. etc.

you can get some tips from the following previous answers:

https://www.researchgate.net/post/Lysis_of_cell_membrane_in_cell_debris_using_RIPA_lysis_buffer_to_extract_membrane-bound_proteins_from_the_cell_debris

Another validation step might be to check for another ion channel that is commonly expressed in HEK 293 cells. If another common ion channel is detected by WB in your lysate, it could be the problem of transfection (or weak expression of the transfected plasmid). If another common ion channel is not detected by WB in your lysate, it would be the protein isolation problem.

Hi Alexander Bontempo

Thanks a lot for your answer,

I am not quite sutre if i got your question right, but no i am just assessing the Ion channel protein WT and mutated and mutation with WT (co-transfected) (it is potassium channel KNa1.1) and the primarry antibody is againest the wt, so it should be positive with wt at least

Please let me know more about what you meant in your question, as i am still quite new in this :)

Hi Mhd Yousuf Yassouf

That is really a great advise, i think i understand your point now, thank you very much i will try again with this tips and hopefully it works.

@sara hawass constitutive gene -> housekeeper gene such as actin b or GAPDH.