Hi Jesse,

In my experience a commercial buffer was not as good for stripping as the home made one from abcam. This is for mild stripping: 15gr Glycine, 10ml of 10% SDS, 1ml Tween, pH it to 2.2 with HCl and then make it up to 1L. In general we strip for 2x10min with enough buffer to well cover the membrane (one membrane per dish). Then we have a few washes in TBS-T for 5-10min. The movement of the buffers has to be really strong so that a wave of buffer goes over the membrane. Abcam also has a home made buffer on their website for stripping proteins that very resistant for stripping. The length of stripping and washing have to be optimised. 30 min completely removed all my proteins. I am not sure what you mean by this statement "The tests of 1° and 2° removal showed very very very reduced signal, so I tried blotting for ERK." To me it implies that not all of your secondary and/or primary Abs were removed, so reprobing would not work.

Why don't you run a sample in all wells of the gel and after detecting your proteins cut the membrane into a few strips and subject them to stripping, different strips for different length of times. Then probe for primary, then secondary and then ECL it. I think this is just as efficient as trying to see whether you removed all your secondary first and then your primary. Stripping length is the most important and first have the standard washing time as per abcam protocol which is 5min. Our method is a little bit different from theirs as we only use TBS-T for washing but we strip at room temperature. We also block again (ie 1hr) after stripping as this prevents background. Removal of your primary is important if you want to reprobe with the same antibody.

You can stain your blot with Ponceau S but it will only give a general idea of how much protein overall is on your membrane. It will be very difficult to tell whether your target protein is removed from the membrane or not with stripping and that is why you can not detect it with your antibodies. Different proteins have differing strength of association with the membrane and your target protein may just be too easy to remove. However, if you do not see much protein on your membrane, then too much protein overall has been removed. Obviously you aim to remove your primary and secondary antibodies. Sometimes a particular target protein-primary Ab association is so strong that you can not remove the primary antibody or when it is removed, it also removes the target protein with it. So try to see which primary Ab is easier to remove and blot for that one first, then strip and blot for the Ab that has the strongest association. I do not see why Ponceau S restaining would affect the membrane.

Tips: Using two different secondary antibodies to detect your proteins may work better once your stripping is optimised. Sometimes some antibodies do not work well with re-probing, making the membranes spotty. Try not to dry out your membranes at any stage.

Hope it helps.

Hi Jesse,

In my experience a commercial buffer was not as good for stripping as the home made one from abcam. This is for mild stripping: 15gr Glycine, 10ml of 10% SDS, 1ml Tween, pH it to 2.2 with HCl and then make it up to 1L. In general we strip for 2x10min with enough buffer to well cover the membrane (one membrane per dish). Then we have a few washes in TBS-T for 5-10min. The movement of the buffers has to be really strong so that a wave of buffer goes over the membrane. Abcam also has a home made buffer on their website for stripping proteins that very resistant for stripping. The length of stripping and washing have to be optimised. 30 min completely removed all my proteins. I am not sure what you mean by this statement "The tests of 1° and 2° removal showed very very very reduced signal, so I tried blotting for ERK." To me it implies that not all of your secondary and/or primary Abs were removed, so reprobing would not work.

Why don't you run a sample in all wells of the gel and after detecting your proteins cut the membrane into a few strips and subject them to stripping, different strips for different length of times. Then probe for primary, then secondary and then ECL it. I think this is just as efficient as trying to see whether you removed all your secondary first and then your primary. Stripping length is the most important and first have the standard washing time as per abcam protocol which is 5min. Our method is a little bit different from theirs as we only use TBS-T for washing but we strip at room temperature. We also block again (ie 1hr) after stripping as this prevents background. Removal of your primary is important if you want to reprobe with the same antibody.

You can stain your blot with Ponceau S but it will only give a general idea of how much protein overall is on your membrane. It will be very difficult to tell whether your target protein is removed from the membrane or not with stripping and that is why you can not detect it with your antibodies. Different proteins have differing strength of association with the membrane and your target protein may just be too easy to remove. However, if you do not see much protein on your membrane, then too much protein overall has been removed. Obviously you aim to remove your primary and secondary antibodies. Sometimes a particular target protein-primary Ab association is so strong that you can not remove the primary antibody or when it is removed, it also removes the target protein with it. So try to see which primary Ab is easier to remove and blot for that one first, then strip and blot for the Ab that has the strongest association. I do not see why Ponceau S restaining would affect the membrane.

Tips: Using two different secondary antibodies to detect your proteins may work better once your stripping is optimised. Sometimes some antibodies do not work well with re-probing, making the membranes spotty. Try not to dry out your membranes at any stage.

Hope it helps.

Hello Jesse,

Frankly I never used the commercially available stripping buffer, so we always work with the home-made one. Here is our standart protocol:

1) incubation in water bath 42 deg (30min) in stripping buffer (100mM 2-ME, 2%SDS, all in PBS );

2) washing in 1xPBS 3 times 10 min rt;

3) proceed with standard blocking

Sometimes when using this protocol, especially when you have a strong reaction some antibodies can still retain on the membrane, so extended incubation in stripping buffer may be needed, but in general it works. Still you can reduce the 2-ME concentration and decrease the incubation temperature to 37 or even to rt.

I can offer you quite the same strategy as Anico suggested. I think you can run the same sample in equal concentration in a number of wells, transfer, stain with Ponceau S to verify that everything is fine. But then, do cut the membrane for strips before detection. Next detect your bands like you do (you should have the same strength of signal everywhere) if not take only the strips with identical strength for further study. Next strip the strips using different protocols, stain with Ponceau S and go on with second staining and detection this would help you to find a suitable stripping procedure. You can also stain your membrane with anti-beta actin ab for control. I should notice that we block no less than 3hr at rt or ON 4 deg C.

Good luck

Alexey

We always had good results with Reblot plus (strong) used as directed (15 min rt with shaking) but we probed for pERK first and then after with more highly expressed proteins and finally with beta actin.

I think the troubleshooting suggested above is also a good control to run! All the best!

Jesse

I have used both Pierce's Restore and the newer Restore Plus. I agree with the stripping approach suggested so far- you test different stripping conditions and see what your blot looks like. I have found a number of protein targets to have been stripped sufficiently by using a 5-10 minute incubation with shaking, followed by three washes of TBS-T for 5 minutes each. i would think this protocol should work for many protein targets. Reduce the incubation to less than 5 minutes if your protein is sensitive to stripping. The Restore Plus is supposed to support stripping up to 4-5 times.

My stripping buffer is:

0.5 M Tris-Cl, pH 6.8 12.5 ml

10% SDS 20 ml

2-Mercaptoethanol 0.7 ml

sH2O 66.8 ml

at 60-70 C for 45 min (with shaking preferably) followed with washing for up to 1 hour ( 4-5 changes of wash buffer) at RT then re-blocking.

Hi Jesse,

What type of membrane are you using? In my experience, PVDF is great for stripping and re-probing, whereas nitrocellulose doesn't work so well. I've successfully used the same Restore buffer that you're using, as well as a home-made glycine-SDS buffer, both with similar results.

It sounds like your problem is not with the stripping itself, since you get very little signal when you test for remaining primary or secondary antibody. I think it would be important to do a Ponceau stain before your initial WB and then again after stripping, to make sure you're not stripping off a lot of the protein in general.

Assuming that there is still plenty of protein on your membrane, then I would focus on the washes. After stripping a membrane I usually wash 4 x 10min TBST in a relatively large volume (so maybe 25mL or so, if my regular volume for antibody or wash is

Using a mild stripping buffer (e.g. 25 mM glycin, 0.1% SDS, 1% Tween-20 at pH 2.0) results in a redetection of previously used primary antibodies quite often. I think with these stripping buffers you can rather destroy the HRP at your secondary AB or maybe strip away the secondary itself than getting rid off the primary one. For more stringent stripping conditions i recommend using an alkaline stripping buffer (100 mM NaOH, 2 % SDS, 0.5 % DTT) and incubating your membrane with it for 30 min at 55°C. For the washing - i guess - its better not to wash too long but change the washing buffer (TBST or TBS) more often. I am using PVDF and (Optitran) NC membranes and for both i have good results in stripping and reprobing without losing my bound proteins.

Hey all- Thanks so much for all the great responses

I thought I had a post in reply to the initial response... what most of you are suggesting makes sense, and makes me wonder if I am actually doing something else incorrectly. I did try a pilot where I ran pERK and then tried stripping at 5 min and 15 min, but next time I'll just try ERK directly. The problem as I see it is that ERK and pERK and the same molecular weight so I can't tell if I'm seeing ERK or pERK. At least, that's how I understand it.

Furthermore, I'm wondering if I'm storing the blots in correctly. Both times I've piloted this, I've been forced to wait a while after scanning the first blot before stripping and trying the second antibody. By this I mean, a week. I understand to keep blots dry (though I've seen varying recommendations out in internetland), but I've been storing them in ddH2O. I'm wondering if this is the problem. Any thoughts on storage of blots for reprobing? Is this my issue?

I'll start trying some of your suggestions and see what works... As I understand it, the procedure and reagents I'm using have been used for pERK and ERK, with a stripping step. (Oh, I'm using PVFD not nitrocellulose.)

Jesse

To distinguish the 2 proteins, try using an antibody that only pick up the pERK in comparison with the one for ERK. Or run a different percentage gel that helps to spread out the 2 proteins a bit on the gel and estimate the position of the band with that of a marker e.g. Pierce Supersignal Enhanced protein ladder.

The blot can be kept for a long time before stripping/reprobing, but I would recommend not in distilled water- use TBS-T and store in the fridge.

Since stripping of membranes allways results in altered signals compared to unstripped detection, I would suggest, that it is much more reliable and also more reproduceable to simply run two gels, or load your samples twice on one gel and cut in half after blotting, probing one half with pERK and the other half with ERK ABs.

Best Kai

Hi Jesse,

It is some better if you strip your membrane right after development, however you can do it later and a week after the first development it is not a best before date, so I don't think it is a problem. There are different recommendations you can find whether to dry or not to dry the PVDF, I heard both works. So if you keep it in PBST or TBST for a week at +4 it is fine. I personally work with nitrocellulose much more frequently and I dry it after transfer and after development, and I can keep it for months in the fridge.

Good luck,

Alexey

Hi Jesse,

As you can see from all the contributions, there are different stripping buffers and protocols and you just have to find, and optimise the one that is best suited for your protein. I prefer not to leave the membranes for a week before stripping but even after a week in TBS-T buffer the signal is fine. I could see that your problem was that both of your bands are about the same size and that is why I suggested using antibodies made in two different species. Then you can be sure that after stripping you are picking up the protein you want and not signal from the protein you previously tested for.

Cheers

Hi Jesse,

Are you using PVDF or nitrocellulose membranes? I have found that PVDF works well whereas nitocellulose does not. Are you stripping right after you develop your blot? If not, try that - the longer you leave the blot, the less likely you will be to get a good signal.

Also, you mentioned that the first time you stripped, that there were still some faint bands - I find this happens no matter what stripping buffer and protocol you use if the signal is particularly strong for that antibody - in that case, I'd suggest you probe with the weakest antibody you plan to use first and then probe with the more robust one second.

Additionally, have you tried increasing the concentration of the antibodies on the second probing? Often the stripping results in a reduction of the robustness of the second signal.

Finally, how old is the stripping buffer? I've had experience with Restore where it worked great one week and not so great the next and the only difference was the freshness of the buffer.

Hope this helps and best of luck!!

Anne

Has anybody tried Immobilon IPFL00010 meant for FITC -2 ab,  for HRP conjugated secondary ? Seems like this membrane doses not even stain with Ponceau?