storing WB blots in PBS-tween - (Nov/12/2009 )

I'm running gel on my proteins total lysates and then transferring to nitrocellulose membrane. I 've heard that you can store the blots in PBS tween for few days if necessary. I've noticed when taking pictures the total absence of any bands for all the preoten antibodies that im using. Is it either because of storing in PBS-T for several days or because of my multiple freezing thawing cycles for the lysates that could change protein conformation?

Some good information on here from a previous post

http://www.protocol-online.org/forums/inde...lulose+membrane

Regarding the numerous freeze-thaw actions

i think is fine as most people use only one lysate for all their blots,

however if some proteases present in the lysate, for example, if the protease inhibitors in your extraction buffer did not work correctly or sufficiently then there could be issue with the degradation of uour proteins each time you thaw the samples

other than this maybe your protein is not present in your lysate at all

the protein is present abundantly as detected from previous blots but my concern is that the dramatic diminishing of signals is either because of storing several days in large volume (300ml) of pbs-t or either because multiple freezing thawing as protein conformation can change

cotchy on Nov 12 2009, 08:11 AM said:

Hi Fred,

It could be because you are storing in in the PBS-T. Sometimes I have this problem when I store certain blots in TTBS for several days. If I re-probe, I can detect the proteins again.

I don't think it would be due to multiple freeze-thaw cycles before running on the blot--initial detection on the blot would be impaired due to this.

regards,

lab rat

thanks i'm leaning towards thinking that too. I think 4 days in PBS-T caused that because the colored markers on the membrane has lost significantly their colors indicating that proteins is might be released from membrane another question that i wish to find answer of: can the primary ab be stored for in the blocking solution at 4 C if so for hwo many days and how many times can it be used? thanks

If your dyes are coming off the membrane, your antibodies may be releasing, too. The bond between target and antibody isn't permanent--ask anyone who has tried to do flow cytometry several days after staining cells. The proteins should be permanently stuck to the membrane.

I recommend using freshly-made antibody in blocking buffer. The lower the concentration, the greater chance of losing your antibody. (It will adhere to the plastic of the tube.) Keep your mAbs in their stock bottles until ready to use. I have used working dilutions that are up to 4 hours old with no ill effects. Use your diluted antibody once.



edited b/c of grammatical error.

Freeze/thaw cycles do damage proteins significantly. I would recommend not going over 4 or 5 thawings of a particular lysate for ANY protein, you are better off aliquoting and thawing each aliquot once than thawing lots of times. This is especially true if you are trying to make any sort of qualitative or quantitative assessment from your westerns.

PBS-T will remove antibodies from blots, this is why it or similar solutions are used for the wash steps in immunodetection. Try storing them in straight PBS or blocking solution to stop the dissociation of the antibody interaction.

i've read that people suggest PBS-T and others PBS whats true and whats not? i will store (in PBS or PBS-T) no longer than O/N even that i've noticed that there times that proteins are maintained well on membrane after 3 or even 4 days and at times there completely gone from membrane