Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

When to wash and how many times?


  • Please log in to reply
4 replies to this topic

#1 Biochem_newbie

Biochem_newbie

    member

  • Active Members
  • Pip
  • 28 posts
0
Neutral

Posted 13 May 2011 - 07:19 AM

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!

#2 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 13 May 2011 - 07:24 AM

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!!

#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,781 posts
132
Excellent

Posted 13 May 2011 - 10:50 AM

we don't wash after blocking. we wash 3x10 minutes after primary and secondary antibodies.
talent does what it can
genius does what it must
i do what i get paid to do

#4 GNANA

GNANA

    GNANA

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 207 posts
9
Neutral

Posted 13 May 2011 - 12:21 PM

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...,
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#5 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,713 posts
398
Excellent

Posted 13 May 2011 - 04:33 PM

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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.