Protein transfer conditions

Original Message
From:Leadin
Date:05/24/2012 15:35:00
So, after performing the transfer shouldn't I rinse with TBST? In fact, should I use TBST any time during the WB? I used a protocol with 3 TBST rinses (3x5min) between almost every step of the WB: between blocking and 1ary Ab, between 1ary and 2ary Ab and between 2ary and revealing buffer, even more, I dissolved Abs in TBST... I didn't know TBST could unattach proteins from the membrane

Using water is only important for destaining after incubating in Ponceau S. Any soultion with salt in it will destain everything. You can use TBST for anything else you want. I use OVDF for transfers. After the transfer, I remove the membrane from the apparatus and let it dry completely. This permanently attaches the protein to the membrane. I then rewet the membrane with methanol, then water, then stain in Ponceau S. With nitocellulose, you can skip the methanol. The important thing is not to have any salt around when staining, or when destaining.

So, after performing the transfer shouldn't I rinse with TBST? In fact, should I use TBST any time during the WB? I used a protocol with 3 TBST rinses (3x5min) between almost every step of the WB: between blocking and 1ary Ab, between 1ary and 2ary Ab and between 2ary and revealing buffer, even more, I dissolved Abs in TBST... I didn't know TBST could unattach proteins from the membrane

Original Message
From:Leadin
Date:05/24/2012 13:26:00
Even if I can't asure you right now the composition of the Ponceau S I'm almost sure it was similar to the one you wrote. What I'm certain is that I don't destain with water but with TBST and maybe I don't wait for 10 minutes but more or less 4-6min; next time I'll keep it longer

thanks

That could be part of the problem. The TBST, or anything with salt in it (like PBS even), will remove the stain from the membrane AND the protein. Water only removes it from the membrane.

Even if I can't asure you right now the composition of the Ponceau S I'm almost sure it was similar to the one you wrote. What I'm certain is that I don't destain with water but with TBST and maybe I don't wait for 10 minutes but more or less 4-6min; next time I'll keep it longer

thanks

Original Message
From:Leadin
Date:05/24/2012 12:26:00
Sorry I wasn't clear enough, I meant I used the manufacturer's recommended volume for the MW marker, for ponceau S I just add some drops to stain all the membrane and observe for some minutes before realizing it won't reveal anything... at the begining I used 2x of the MW marker manufacturer's recommended volume with similar results. Since the last time I wrote in this forum I checked all the steps and modified anything suspicious to be wrong: transfer buffer, gel casting, usage of cooler unit and stirr bar... so I guess everything is well done now; next step may be using prestained MW marker.

Thanks

Thanks for clarifying. It's a little odd that the markers don't show up. Just for a bit more clarification: Ponceau S at 0.2% (w/v) in 5% (v/v) acetic acid; stain the blot for 10 min with agitation, destain with water. Is that how you do it? All but the very darkest bands won't be visible until after destaining with water.

Sorry I wasn't clear enough, I meant I used the manufacturer's recommended volume for the MW marker, for ponceau S I just add some drops to stain all the membrane and observe for some minutes before realizing it won't reveal anything... at the begining I used 2x of the MW marker manufacturer's recommended volume with similar results. Since the last time I wrote in this forum I checked all the steps and modified anything suspicious to be wrong: transfer buffer, gel casting, usage of cooler unit and stirr bar... so I guess everything is well done now; next step may be using prestained MW marker.

Thanks

Original Message
From:Leadin
Date:05/24/2012 07:33:00
I'm using HRP conjugated Antibodies and DAB based revealing buffer with 0.03%NiCl2.

Is it usual not to get the MW marker signal after adding Ponceau S? I use the manufacturer recommended volume. Once again, would a longer transfer time improve the ammount of protein transfered? I performed a 1 hour transference.

Thanks

The detection limit for Ponceau S is about 250 ng/band. This amount is somewhat variable depending on the specific protein being stained.
I use the manufacturer recommended volume. What detection method is this recommended volume intended for? It may work for Coomassie staining of the gel, for example, but be insufficient for Ponceau S on a blot. You could try a titration of the markers, starting with the recommended volume and also 2x and 4x volume.

Have you consulted the instruction manual for the transfer apparatus to make sure you are doing everything according to the recommendations? For example: are you using the correct buffer (without pH adjustment); are you starting with cold transfer buffer and using the cooling block; are you pre-equilibrating the gel in transfer buffer; are you using nitrocellulose with 0.2 µm pore size (0.45 µm will also work but the smaller pore size will catch more of the proteins)?

If everything is done according to the recommendations, 1 h at 100V should be fine. If you want to go longer, you can do it overnight, at 30V, in the cold room. Going longer than 1 h at 100V risks overheating.

I'm using HRP conjugated Antibodies and DAB based revealing buffer with 0.03%NiCl2.

Is it usual not to get the MW marker signal after adding Ponceau S? I use the manufacturer recommended volume. Once again, would a longer transfer time improve the ammount of protein transfered? I performed a 1 hour transference.

Thanks

Original Message
From:Leadin
Date:05/24/2012 02:04:00
Hi again,

after some arrangements I performed another WB and even when Ponceau S didn't reveal proteins (not even the protein marker) I decided to go on and tried to detect actine, and this was the result :
WesternBlot

The point is that I got a clearly defined band which from my point of view is not susceptible to be an artefact, but I only got that band and no previous Ponceau S signal... I guess that the protein transference worked but do you think I should increase the time for protein transfer to get better results (and hopefully get a Ponceau S signal to see the protein marker)?

Thank you

Ponceau S is much less sensitive than a WB. So, you could still have proteins on your blot but not see any with Ponceau S. I would suggest running a dilution series of some complex protein mixture, like a cell lysate, blotting the gel and then staining with Ponceau S. For a total cell lysate, you could try running 40 µg in one lane and then 2-fold dilutions in the others until you use the entire gel. So, you would have 40, 20, 10 etc µg per lane.

Another good way to check for protein transfer is with prestained MW markers. This will give you immediate feedback as to whether your proteins transferred or not.

What detection system are you using on your WB?

Hi again,

after some arrangements I performed another WB and even when Ponceau S didn't reveal proteins (not even the protein marker) I decided to go on and tried to detect actine, and this was the result :
WesternBlot

The point is that I got a clearly defined band which from my point of view is not susceptible to be an artefact, but I only got that band and no previous Ponceau S signal... I guess that the protein transference worked but do you think I should increase the time for protein transfer to get better results (and hopefully get a Ponceau S signal to see the protein marker)?

Thank you

Hi all,
I'm trying to perform a Western Blot of proteins purified from tissue in a weight range between 15 and 95kDa with a SDS-PAGE at 12%. The main problem to date has been during the protein transfer to nitrocellulose membrane; I use MiniTrans-Blot from Bio-Rad wet transfer system, the only treatment I apply to membrane is soaking it in cold transfer buffer. I tryed 100V for 1 hour and it looked as if proteins had trespassed the membrane (transfer buffer got blue colour), when I tried to die the membrane with Ponceau S not even the protein marker was coloured so I stopped the WB protocol. Which transfer conditions would you recommend to me?
Thank you

First of all, did you follow the instructions as described in the instruction manual (link)?

I haven't used that system for several years but it worked fine. I now use the Bio-Rad semi-dry system and like it very much. Check over the instructions to make sure you are doing everything correctly. Pay particular attention to the buffer recipe, electrode direction, cooling system, membrane pretreatment, and recommended voltage/time/and mA readings.

Hi all,

I'm trying to perform a Western Blot of proteins purified from tissue in a weight range between 15 and 95kDa with a SDS-PAGE at 12%. The main problem to date has been during the protein transfer to nitrocellulose membrane; I use MiniTrans-Blot from Bio-Rad wet transfer system, the only treatment I apply to membrane is soaking it in cold transfer buffer. I tryed 100V for 1 hour and it looked as if proteins had trespassed the membrane (transfer buffer got blue colour), when I tried to die the membrane with Ponceau S not even the protein marker was coloured so I stopped the WB protocol. Which transfer conditions would you recommend to me?

Thank you