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Actually I have ordered the anti integrin beta 5 (ab15459) to used in Immunocytochemistry and I want to start with your protocol but I have confused in understanding the blocking step. As it is shown in your protocol that cells must be incubated in 1 % BSA/ 10% normal goat serum/0.3 M glycine in 0.1 % PBS-Tween. Do you mean to choose one of them or as a compound in 100 ml of PBS. Also I want to know what is the perfect incubation temperature??
Asked on Feb 16 2012
Thank you for your inquiry.
Yes, the blocking buffer that was used to generate that image was a combination of 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The permeabilization and blocking steps were done at the same time this way. You can perform this step at room temperature. This is the protocol we've used in house.
We have heard that a customer tried separating out the steps and it worked:
Yes - 0.1 % Triton X-100
BSA as blocking agent for 30 mins · Concentration: 2 % · Temperature: 25°C
I hope this information helps. Please contact us with any other questions.
Answered on Feb 16 2012