6 replies to this topic

[Tai]

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

[Dr Teeth]

Tai, on Sep 9 2009, 07:05 AM, said:

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.

[madrius1]

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

[Dr Teeth]

madrius1, on Sep 9 2009, 11:23 AM, said:

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

We store ours at 4°C.

[miBunny]

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  

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.

[Tai]

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%

[Dr Teeth]

Tai, on Sep 10 2009, 02:30 PM, said:

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%