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
- - - - -

if antibody solution could be used again?


  • Please log in to reply
8 replies to this topic

#1 lufang

lufang

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 24 March 2009 - 03:34 AM

The first-antibody in blocking buffer (PBS with 3% BSA) and the second-antibody in blocking buffer (PBS with 5% nonfat dried milk), both of them were stored at 4℃ for one month, if i can use them for western blotting again? thanks.

#2 rkay447

rkay447

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 180 posts
20
Excellent

Posted 24 March 2009 - 03:54 AM

As long as you don't see any bacterial growth, the primary antibody should be fine. If you do see growth (look for the pellet at the bottom of the tube) you may be able to filter the dilution and still have a usable dilution but the bacteria may have been able to lower the pH and destroy the antibody. I never save or reuse secondaries so I'm not sure how that will work if you try. If you want to save you primary antibody dilutions you should add 0.05% sodium azide. This will prevent the bacterial growth but kills the HRP on the secondary. Hence why most people don't reuse the secondary.

In fact, rather than stripping a membrane, I use NaAz (1% for 1 hour) if I want to reprobe with an antibody of a different species. This destroys the HRP (and signal) from the first antibody and then I can reprobe without any signal from the first antibody.

#3 mdfenko

mdfenko

    an elder

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

Posted 25 March 2009 - 06:27 AM

the milk with your secondary may have gone bad and may require replacing.
talent does what it can
genius does what it must
i do what i get paid to do

#4 mastermi

mastermi

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 82 posts
0
Neutral

Posted 26 March 2009 - 04:40 AM

I store my primary antibodies after use at -20C and reuse them 5-8 times! At -20C you won't have any problems with bacterial growth. Works perfectly.


If you want to save you primary antibody dilutions you should add 0.05% sodium azide. This will prevent the bacterial growth but kills the HRP on the secondary. Hence why most people don't reuse the secondary.

In fact, rather than stripping a membrane, I use NaAz (1% for 1 hour) if I want to reprobe with an antibody of a different species. This destroys the HRP (and signal) from the first antibody and then I can reprobe without any signal from the first antibody.


NaAz is quite toxic. How do you handle your antibody, your membrane and everything that was in contact with it?
Cause when I'm working with NaAz, I dispose everything as hazardous waste.

#5 Dr Teeth

Dr Teeth

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 221 posts
1
Neutral

Posted 26 March 2009 - 05:01 AM

We reuse our antibody solutions for months unless bacterial growth occurs. The trick is to not use milk. By diluting our antibodies in TBST rather than milk and storing at 4C, they last for months. Comparative blots run months apart show similar staining for all of our antibodies. Non-specific binding even appears to decrease over time slightly.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#6 MaggieRoara

MaggieRoara

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 30 March 2009 - 01:37 AM

We reuse our antibody solutions for months unless bacterial growth occurs. The trick is to not use milk. By diluting our antibodies in TBST rather than milk and storing at 4C, they last for months. Comparative blots run months apart show similar staining for all of our antibodies. Non-specific binding even appears to decrease over time slightly.



Just wondering, Maybe if you filtered before storage,i.e. its sterile. it should last longer right even with milk

#7 mdfenko

mdfenko

    an elder

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

Posted 30 March 2009 - 11:11 AM

We reuse our antibody solutions for months unless bacterial growth occurs. The trick is to not use milk. By diluting our antibodies in TBST rather than milk and storing at 4C, they last for months. Comparative blots run months apart show similar staining for all of our antibodies. Non-specific binding even appears to decrease over time slightly.



Just wondering, Maybe if you filtered before storage,i.e. its sterile. it should last longer right even with milk

even properly handled and stored, milk doesn't last long (unless it is frozen).
talent does what it can
genius does what it must
i do what i get paid to do

#8 MaggieRoara

MaggieRoara

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 31 March 2009 - 05:40 PM

We reuse our antibody solutions for months unless bacterial growth occurs. The trick is to not use milk. By diluting our antibodies in TBST rather than milk and storing at 4C, they last for months. Comparative blots run months apart show similar staining for all of our antibodies. Non-specific binding even appears to decrease over time slightly.



Just wondering, Maybe if you filtered before storage,i.e. its sterile. it should last longer right even with milk

even properly handled and stored, milk doesn't last long (unless it is frozen).



When you do this, how many times do you usually freeze and thaw before its becomes non-viable?

#9 mdfenko

mdfenko

    an elder

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

Posted 06 April 2009 - 10:14 AM

When you do this, how many times do you usually freeze and thaw before its becomes non-viable?

we don't use milk for that reason. we use bsa and/or serum when we prepare our antibody solutions. even so, the proteins can (and do) go "bad" over time (just smell the solution, you'll know when).

as for how many freeze/thaw cycles the solution can withstand, you may have to determine that for your antibody preparation.
talent does what it can
genius does what it must
i do what i get paid to do




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

©1999-2013 Protocol Online, All rights reserved.