4 replies to this topic

[Biochem_newbie]

Hi there

I would like to get some advice on the washing steps for Western Blot.
I have read that blocking overnight is a good recommendation to decrease the non-specific binding issue.
Also, I have read that washing after blocking (before treatment with 1st Ab) is a good idea, washing before 2nd Ab, and washing after 2nd Ab.

I have been trying these out but I was wondering if anyone had any experience with the duration and the number of times the wash should be exchanged?
I have been doing 2 times 30 mins for before 1st Ab, and after 1st ab, and 6 times 10 mins for after 2nd Ab.

Does anyone have any suggestions regarding washing?

Would very much appreciate it

Thank you!

[almost a doctor]

I think is better to do more short washes, than less long washes.  

2x 30min seems excesive after blocking (even after the 1st Ab), I'll change that to 2 or 3x 5-10min washes.  For the final washes, 6 also seems a bit too much to me, but all depends on your particular experiments and how "dirty" your blot might be. Western blot is very much sample and antibody dependet, so whatever works best for you, just stick to that!!

[mdfenko]

we don't wash after blocking. we wash 3x10 minutes after primary and secondary antibodies.

[GNANA]

i have never done or heard abt washing after the blot being blocked, i usually do washes after 1' and 2' maximum 5 washes of 5 min each...,

[bob1]

I do 4 x 5 min washes after primary and secondary... too much washing or too long washes will lead to weak signal.