hello,everyone.
I need to solve this problem in western blot , i did the cell lysis by NP40 then protein assay kit to quantification the protein . i load same amount of protein 70 ug but different vol for exp:
HCT8 con 13.4 ug/ul protein vol 3.7 ul 2x Lammili 3.7 ul
HCT116 con 10.2 ug/ul protein vol 4.9 ul 2x Lammili 4.9 ul
then i repeated with same amount 70 ug and same volume i get same result.
Waiting for your helps,Thank you .
Hi,
firstly I think you load 50 microg per wells.
Secondly :have you start again your quantification? Did you extract the proteins with the same protocol for every cells lines? did you store them in the same conditions? to sum up : Is your complete procedure common/the same for all your cell lines?
Thirdly, if you have done well your extraction, the thing is that you work with different cell lines. The expression level of a protein even if an ubiquitously expressed one like the Beta-Actine can change according different cells lines.
for this final point you can check your western blot procedure with other proteins like GAPDH.
All the best,
Greg
Hello Amal
I agree with contribution of Gregory.
Have you use protease inhibitors in you lysis buffer?.
Best regards
Grégory Fettweis , thank you very much for your help .
Yes, sorry I load 5o ug.
before every experiment I did the quantification for the samples and i used same condition for all cell line from extraction to store the sample ( lysis buffer I follow the kit instructions) , except for ccd841 I didn't have stock so I used old sample.
I will take your advice in consideration, for trying other proteins.
Thank you
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