14 replies to this topic

[jiro_killua]

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?

[WHR]

jiro_killua, on Mar 8 2009, 08:33 PM, said:

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.

[little mouse]

Keep it few days at 4°C or add Na azide (not with HRP-conjugated antibodies) or freeze.
However be careful, some antibodies can lose activity after freezing.

[little mouse]

and about the difference between BSA and milk. It's more a question of quality of blocking. you will choose between both depending on the intensity of the band and the background. I saw no difference between both when I was keeping my antibodies at 4°C or freezing them.

[Curtis]

we do not keep antibody in non-fat milk or BSA or even TBST.

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.

[yobou]

some antibodies are unstable if diluted in TBST. For example in the datasheet of sigma antibodies  its mentioned that the antibody is stable for up 16 hrs (at 4 oC) when diluted in TBST.

[Tfal]

I do reuse 1st ABs a couple of times (rarely more) if I incubate at 4 C (ON). I keep it at 4 C if it is to be reused within a couple of weeks but past that I freeze them. Yes freezing/defreezing might alter some ABs but it's hard to make sure that nothing has grown in only a couple of ml of BSA or even more so in milk.

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.

[minylim]

I've stored primary antibody in milk at -20c and sometimes white precipitation of milk was found after thawed it (and blot didn't work). FYI...

[laurequillo]

We dilute our antibodies in TTBS with milk, and we keep them at 4ºC. Each antibody is different; i.e. anti flag from sigma last more than two month in dilution, but anti HA from roche just two weeks. Normally I keep the secondary antibodies just 10 days in dilution.

[Feelcontraire]

jiro_killua, on Mar 8 2009, 09:33 PM, said:

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

[medchemgirl]

Can you re-use secondary Abs-HRP @4C in TBS-T?

[Feelcontraire]

medchemgirl, on Nov 19 2009, 12:16 PM, said:

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.

[medchemgirl]

Uuuuuuu That sounds nice!!! Thank you!

Feelcontraire, on Nov 19 2009, 07:41 PM, said:

medchemgirl, on Nov 19 2009, 12:16 PM, said:

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.

[eldon]

we've used primary antibodies over and over ( at least 5-6 times before noticeable loss in signal). we never store in milk or bsa and don't like using azide. we block our membranes first with milk or bsa...rinse 3X with 1XTTBS..then add the antibody diluted appropriately in 1XTTBS. after incubation we collect the antibody solution and filter sterilize it into a 50ml falcon tube and store at 4C. we then re-block the membrane briefly and proceed with secondary.

saves a lot of money.

[ajxutao7]

antibodies are so expensive. usually, I will reuse the antibody within 1-2 weeks, have tried antibodies in 2% skim milk, three weeks old, 4 degree, it still worked but less 10 times sensitive as fresh one. after 1 month, it didn't work at all.

hope it helps