
Tips on re-use of primary antibody in Western blot
#1
Posted 08 March 2009 - 08:33 PM
If I want to reuse antibody for western blot, does it matter what I initially dilute it in?
normally, I add the antibody to 5% non-fat milk in TBST
but then can I keep the antibody in milk at 4C for long?
Some ppl mentioned 5% BSA in TBST, I wonder would that make any difference?
#2
Posted 08 March 2009 - 10:34 PM
quick question:
If I want to reuse antibody for western blot, does it matter what I initially dilute it in?
normally, I add the antibody to 5% non-fat milk in TBST
but then can I keep the antibody in milk at 4C for long?
Some ppl mentioned 5% BSA in TBST, I wonder would that make any difference?
It would be fine if nothing grows in it.
#3
Posted 09 March 2009 - 12:17 AM
However be careful, some antibodies can lose activity after freezing.
#4
Posted 09 March 2009 - 12:28 AM
#5
Posted 14 March 2009 - 07:16 AM
in our lab we always dilute our antibodies (both primary and secondary) in 1xTBS....I personally re-use my antibodies 5-6 times, if I feel the bands are fading away I just add a little more to the same solution. but it would be better to make a new solution if you feel the bands are not good enough.
we all get sharp and clear bands on our PVDF membranes.we have had no problem so far but we know it is NOT an internationally standard method.
#6
Posted 27 June 2009 - 09:03 PM
#7
Posted 08 July 2009 - 07:58 AM
I don't reuse 2nd AB's since I always incubate at RT, I dilute them much more and they tend to be less expensive.
#8
Posted 21 August 2009 - 11:30 AM
#9
Posted 25 September 2009 - 03:42 AM
"This is SPARTA!"
"I´m the goddamn batman"
#10
Posted 18 November 2009 - 08:22 AM
quick question:
If I want to reuse antibody for western blot, does it matter what I initially dilute it in?
normally, I add the antibody to 5% non-fat milk in TBST
but then can I keep the antibody in milk at 4C for long?
Some ppl mentioned 5% BSA in TBST, I wonder would that make any difference?
Usually it is not recommended to freeze-thaw antibodies as it may lead to a lost of conformation.
It isn't also recommended to leave the antibodies at 4ºC without at least 0,1-1% of other protein like BSA, to scavenge protein modification and adsortion to the tube walls.
That been said is common to find that under this conditions the antibody is still working perfectly.
If you plan to leave your antibody at 4º:
Mandatory:ad 0,05-0,1% Thimerosal to avoid fungal grow.
Basic:the more concentrated the antibody the more stable it´ll be withou much extra stuff, I think that till 0,2mg/ml is okay, usually 1mg/ml
Not recommended: milk, milk gets bad very fast, in a few days.
Recommended: dilute in 0,1-3% BSA for reasons above.
Usual-good idea: add some tween20 to avoid adhesion to the tube wall, and maybe also intermolecular adhesion.
Stabilizing solution formulation: they usually contain thimerosal, tween20, bsa and/or casein, sometimes D-Mannitol and PVA.
If you prentend to predilute secondary antibodies that carry HRP you should do it in 1%PVA (130-180k).
Aliquots and predilutions can be performed in 50%Glicerine and stored at -20º. This keeps kinetics very low while avoiding freezing. Some glicerine in your final dilution is ok, no influence, but don't pretend to incubate with 50% glicerine, difusion and binding may be reduced.
In fact, PVA is a great instant PVDF block.
After all this, what I usually do is either freeze and store at -20
#11
Posted 19 November 2009 - 11:16 AM
#12
Posted 19 November 2009 - 04:41 PM
Can you re-use secondary Abs-HRP @4C in TBS-T?
Long term, nope, HRP breaks down very fast in water. During 1-3 days probably, but activity will start to decline.
Reusing your secondary can help you to remove unspecific binding if you have some.
If you pretend to prepare a replenish stock or something like this, it should be in 1% PVA to stabilize de HRP.
The good thing about this is that such a high amount of PVA will give very clean background.
#13
Posted 02 December 2009 - 02:34 PM
Can you re-use secondary Abs-HRP @4C in TBS-T?
Long term, nope, HRP breaks down very fast in water. During 1-3 days probably, but activity will start to decline.
Reusing your secondary can help you to remove unspecific binding if you have some.
If you pretend to prepare a replenish stock or something like this, it should be in 1% PVA to stabilize de HRP.
The good thing about this is that such a high amount of PVA will give very clean background.
#14
Posted 02 December 2009 - 03:51 PM
saves a lot of money.
#15
Posted 10 May 2011 - 10:09 PM
hope it helps