Hello everyone. I hope someone could give me a suggestion and tips&tricks.
I have been trying to detect p-eNOS on HUVECs cell line using WB and got nothing (literally my visualisation was clear) for several times. My antibody is p-eNOS (Ser1177) Rabbit polyclonal antibody from BEYOTIME and when the assay I did 1:1000 dilution.
The WB protocol I did was common. Do I need to add something (when the cell culture or lysis process) to induce p-eNOS expression?
Thank you!
Hi Andy,
what about your reference gene? does it has a sharp band on the membrane? what type of secondary antibody do you use?
Hi Andy,
Many things can influence detection of p-eNOS by WB. For beginning make sure that you have phosphatase inhibitors when you are preparing cell lysates. Also, try to counter detect eNOS on the same blot to make sure that you have eNOS protein there.
Best of luck!
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