Hello,
I am trying to detect PAK1, PAK2 in cell lysates from HL60 cell line in western-blot.
I tried to go for 50ug of protein but I just had a very tiny little band.
I thought 50 ug of prot would be enough...
I use NUPAGE system (gradient gel = 4-12% Bis tris), wet transfer (35 V for 2 hours). Blocking using TBS blocking solution (LICOR-Odyssey), Ab used = PAK1= 2602 from CST, PAK2= 2608 from CST over night, anti rabbit 680 from LICOR.
Does the proliferation state of this cells car change dramatically the expression level of this proteins?
Do you have any tips maybe to increase the intensity of my bands?
(Also, can 't detect c-RAF...).
If you have previously run this kind of experiments can you please write the details of your western blot?
Many thanks in advance,
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