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Can primary antibody be reused ?


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6 replies to this topic

#1 Tai

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Posted 09 September 2009 - 03:05 AM

Hi all

I always prepare the P-antibodies in the TBST + 3% skim milk at 1:1000 p-ab and incubate PVDF membrane about 1.5h at RT,
or sometimes I incubate membrane over night at 4 C

Does anyone has ever tried to reuse this p-ab solution for another WB ?
If can do, what is the good way to store the p-ab solution or how long it can be kept ?

lasty, what is the good internal control, if not count B-actin, for human fibroblast because my interested proteins is about 43 kDa ?

any suggestion will be appreciated
/Tai
I h..ve ne..er finish.. ...ny thi .....

#2 Dr Teeth

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Posted 09 September 2009 - 05:45 AM

Hi all

I always prepare the P-antibodies in the TBST + 3% skim milk at 1:1000 p-ab and incubate PVDF membrane about 1.5h at RT,
or sometimes I incubate membrane over night at 4 C

Does anyone has ever tried to reuse this p-ab solution for another WB ?
If can do, what is the good way to store the p-ab solution or how long it can be kept ?

lasty, what is the good internal control, if not count B-actin, for human fibroblast because my interested proteins is about 43 kDa ?

any suggestion will be appreciated
/Tai



You can reuse your primary antibody solution a few times over a short time period (I'd say less than a week or so since the milk will start to turn). If you leave the milk out (i.e. prepare your primary solution in TBST alone, or with BSA), you can reuse this solution for months. We do this regularly for dozens of different antibodies, and have even noticed a reduction in background over time when reusing these primary solutions, with no loss in target signal. Also, don't include sodium azide for your stored solutions, since it inhibits HRP.
For another internal control, try RNA polymerase II.

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

#3 madrius1

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Posted 09 September 2009 - 07:23 AM

Some Ab tolerate very well freeze-thaw cycles. We routinely freeze the Ab solution, and reuse them afterwards.

#4 Dr Teeth

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Posted 09 September 2009 - 07:26 AM

Some Ab tolerate very well freeze-thaw cycles. We routinely freeze the Ab solution, and reuse them afterwards.


We store ours at 4C.

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

#5 miBunny

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Posted 09 September 2009 - 05:26 PM

Our observation (not rigorously tested!) was that antibodies that oame in glycerol solutions do significantly poorer when reused then antibodies that are stored frozen in an aqueous solution or are stored at 4 degrees. I confess that the data points are few and that the proper controls were not included :huh:

Anyone else have any comments on this??? Just curious!

I second the background getting better with a lot of the antibodies. Erk always seems to give the best results on use number 2.

#6 Tai

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Posted 10 September 2009 - 10:30 AM

thank you everyone for all comments,

btw, how many % of BSA can be used for p-ab?
Found one Google said 1% somebody said 5%
I h..ve ne..er finish.. ...ny thi .....

#7 Dr Teeth

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Posted 10 September 2009 - 11:27 AM

thank you everyone for all comments,

btw, how many % of BSA can be used for p-ab?
Found one Google said 1% somebody said 5%


either, I'd use 5%

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




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