Membrane Storing

[Arastho]

Originally Posted by 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.

Hi!
Yes, it is my first time but we tested the Ab and o.n. we got better results.
5 membranes means 5 gels and 14x5 samples and that's something that we don't have. (Equipment and samples)
We are doing a stimulation assay during time, so we have different Time Points...

I'm running now the first Ab. At the end of the week I will say something.
Stay tune

[Arastho]

Originally Posted by 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.

If you have different bands with different (or equal) secundary antibodies you don't need to strip.
I just strip the membrane when I need to see bands with the same size and if they are incubated with same secondary.

Strip wasn't the cause of my signal lost. I think that was the drying.
In fact, I re-test and happens the same.
Thanks

[Arastho]

Originally Posted by 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.

Huummm...Nice Sugestion...
I will try that...

[Arastho]

Hi again,

In conclusion: 4ºC cover in sterile PBS only. Is the better solution for storing for a few days.
With TBS-T a lost my signal 3 days after...

[Mad Scientist]

I've read that you need to dip your membrane into methanol before continue if reblot it if you've dried it out for storage at -20. I've never done this, but maybe that would help. I've just kept blots in buffer at 4 degrees between blots...mind you I've never stored a blot longer than 1 week.
I find I have to strip and block between wetsrns of the same membrane to get good results.