3 replies to this topic

[Flavia]

Hi,

I would like to know if this is valid:

I used a primary antibody once and the signal was very good. So I tried to re-use the same antibody in other membrane (I added a little bit more of the antibody at the second time). However, this time the signal didn´t work out.

So, could I keep the membrana in TBST at 4°C until the new antibody arrive and then re-use the membrane?! (I don´t have lots os sample to re-do it).

Thanks a lot

Flávia

[rkay447]

Flavia, on Apr 22 2010, 08:15 AM, said:

Hi,

I would like to know if this is valid:

I used a primary antibody once and the signal was very good. So I tried to re-use the same antibody in other membrane (I added a little bit more of the antibody at the second time). However, this time the signal didn´t work out.

So, could I keep the membrana in TBST at 4°C until the new antibody arrive and then re-use the membrane?! (I don´t have lots os sample to re-do it).

Thanks a lot

Flávia

Sure.  You can keep membranes indefinitely but if you are going to go longer than about one week in TBST I highly recommend you add 0.05% sodium azide to prevent bacterial growth. You should always hang onto membranes from experiments that worked.  I dry my membranes overnight between two sheets of whatman paper on my bench and then seal in plastic.  I tape this into my notebook next to the films and then, if later I want to look at something else, I just rehydrate the membrane and probe as usual.  It's good to hang onto these as you never know when you'll get a great idea or if a reviewer asks for you to look at something.  This way you don't have to redo the entire experiment, just get the original membrane!

[cellgene]

does not the proteins on the membrane get degraded after sometime?
my supervisor had told me that we could use the membranes about one week if we keep at 4degrees.

Edited by cellgene, 25 April 2010 - 12:13 PM.

[rkay447]

cellgene, on Apr 25 2010, 02:12 PM, said:

does not the proteins on the membrane get degraded after sometime?
my supervisor had told me that we could use the membranes about one week if we keep at 4degrees.

I've never heard of this, nor have I ever seen this problem.  I've used membranes for months without any problem.  Plus, I've rehydrated dried membranes over one year later and been able to get good signals.  It might be the type of membrane you use, however.  I almost exclusively use PVDF and am not as familiar with nitrocellulose issues.  I'm just not sure what would make the protein degrade since everything is denatured, separated and fixed onto the membrane.  What your supervisor is potentially thinking of is bacterial contamination which will permanently destroy a membrane.  Hence my suggestion that you add a little NaAz to prevents this.  Hmm.. I'll have to ask around and see if anyone else has heard of this problem.  I'll get back to you if I find anything relevant.