Membrane Storing

[Arastho]

Hello Everyone!

I'm new in "manually" Western Blots. So I need some sugestions from you all.

What could be wrong in this sequence?

After transfer:

Membrane Block
Primary Ab
Wash (5x 10 min TBST)
Secondary Ab
Wash (5x 10 min TBST)
Revelation: Good Signal
Store at -20ºC dry

2 days after (weekend)

Wash (5x 10 min TBST) or (just wash 1x PBST)
Primary Ab (GADPH)
Wash (5x 10 min TBST)
Secondary Ab
Wash (5x 10 min TBST)
Revelation: No Signal

I normally do not strip the membrane because the bands that I'm looking for are completelly diferent bands.

But my main problem now is that I will try to do 5Ab diferents, it means 1Ab p/day because I incubate primary Ab o.n. but I will need to store the membrane 2 days (over the weekend).

Any ideas for the store and re-start Monday?

Thanks

PS - I'm talking about nitrocellulose membranes

[jasmith456]

I didn't think you were supposed to dry nitrocellulose membranes. I believe you can leave the membrane in a small amount of TBS or PBS at 4*C for a few days without a problem.
Perhaps you can leave the largest protein for last, since it may be the least likely to unbind the membrane.
Good luck!

[Tfal]

I think that as long you make your membrane completely dry, you can freeze and defreeze (-20) without problem.
I was also told that if it for a short period , you can keep it in TBS (without tween at 4 C or dry at RT.
I think blocking again for each reuse might be something to try and keep on washing extensively like you say you do.

By the way, what's your 1st primary? See how your secondaries might interact.

Good luck
Tfal

[Tfal]

Ho ya... do you fix you membrane (.05% glutaraldehyde) before first block (normal wash in between)?

Tfal

[Arastho]

Originally Posted by Tfal

I think that as long you make your membrane completely dry, you can freeze and defreeze (-20) without problem.
I was also told that if it for a short period , you can keep it in TBS (without tween at 4 C or dry at RT.
I think blocking again for each reuse might be something to try and keep on washing extensively like you say you do.

By the way, what's your 1st primary? See how your secondaries might interact.

Good luck
Tfal

Hi!
I've been trying some treatments to the membranes.
In fact, drying the membrane is completelly out of question. I try RT, -20 and I lose always the signal!

My best strategy until now is leave under PBS. I also tested PBS+T (someone told me that could avoid contaminations but also could remove some proteins in a long time assay.)

Secondaries interaction?? Could you explain?

[Arastho]

Originally Posted by Tfal

Ho ya... do you fix you membrane (.05% glutaraldehyde) before first block (normal wash in between)?

Tfal

No. What is your idea?

[Arastho]

Originally Posted by Tfal

I think that as long you make your membrane completely dry, you can freeze and defreeze (-20) without problem.
I was also told that if it for a short period , you can keep it in TBS (without tween at 4 C or dry at RT.
I think blocking again for each reuse might be something to try and keep on washing extensively like you say you do.

By the way, what's your 1st primary? See how your secondaries might interact.

Good luck
Tfal

Hi!

From my experience in last days completelly dry the membrane is out of question. I try 4ºC, -20ºC, RT and I lose signal. Faster in -20ºC.

Even not drying the membrane, I lose signal in -20ºC.

In 4ºC PBS could be the solution. PBS-T also but someone told me that TWEEN is good for avoin contaminations but in a long time exposure could remove some protein.

I will tell something soon...

Antibodie interaction? Could you explain?

[RAGO]

Is this the first time you are running this way? You may be able to increase your AB titer and incubate less time - most antibodies today are specific and clean enough that 1-3 hour incubations are plenty. Check with the antibody producer, unless you are using in-house generated antibodies. It may also be easier just to run 5 membranes simultaneously. If you run your protein on the PAGE, then you can cut your membrane and incubate each strip is separate troughs for each antibody.

[RAGO]

You can dry membranes and store them laying on dry blotting paper in a petri dish at 4C. We had one that was stored this way for 5 months and worked fine.

[Anastasia_S]

I don't seem to detect my actin, if I don't strip the membrane from the other Ab I used before that. (Store it at 4 C in TBS). So maybe u too, may have to strip it.