Hi Justin,

I prepare my stripping buffer, the recipe is: 1.5g glycine, 0.1g SDS, 1ml Tween 20, add 100ml di water, pH 2.2.

I also have to strip my nitrocellulose membrane 5-6 times ( 10 minutes) but to avoid loosing the proteins on the membranes i cut my membrane according to the molecular weight. so if i have a proteins with a MW 100kd and above and the others are 70kd and below, i would cut my membrane somewhere in between ( by using MW marker). this way you avoid loosing proteins of your interest.

Hope this would help

Hi Justin,

I used the restore western blot stripping buffer by Thermo Scientific. It worked well for me. I used to incubate my blot with the stripping buffer for 20 to 25 minutes at room temperature to strip off the antibodies (even though the recommended time is 5 to 15 minutes, in my experience, it takes about 20 to 25 minutes for good removal of the prior signal. But I usually don't go over 25 minutes). Here is the product link http://www.piercenet.com/browse.cfm?fldID=02040210 in case you want to order it. Good luck!

Hi Justin,

I prepare my stripping buffer, the recipe is: 1.5g glycine, 0.1g SDS, 1ml Tween 20, add 100ml di water, pH 2.2.

I also have to strip my nitrocellulose membrane 5-6 times ( 10 minutes) but to avoid loosing the proteins on the membranes i cut my membrane according to the molecular weight. so if i have a proteins with a MW 100kd and above and the others are 70kd and below, i would cut my membrane somewhere in between ( by using MW marker). this way you avoid loosing proteins of your interest.

Hope this would help

Hi Justin,

the acetic buffer works quite good. But, you should avoid SDS. Why: SDS denatures your protein immediately. If you would like to have an antibody from the membrane which is still active, don't use it. The low pH (2.2 0.01 mol/L Gylcin/HCl buffer) dissolves the binding anyhow. Time as described above. After the incubation with this acetic buffer you need a neutralization to neutral pH. Just make a short titration to learn how much 0.1 mol/L Tris you need to neutralize a distinct amount of your acetic buffer.

Good luck.

Hi Justin,

I use 62.5 mM Tris-HCl pH 6.7, 100 mM b-mercaptoethanol, 2% SDS. 30 minutes at 50C with occasional agitation. However, I use PVDF as I found in the past that this type of membrane held up better when reprobing several times. Also, I have found that success depends a lot on the order in which the membrane is probed, i.e. I have some antibodies that absolutely will not work after stripping and some that work no matter what.

Good Luck!

anything with b-mercaptoethanol is effective but also messy and potentially harmful to health. I use a low pH (pH = 2.0) Glycine solution with SDS. (25mM glycine, 1% SDS). 30-60 minutes works well for most proteins. (harder for the more abundant proteins). It is easy to make, inexpensive and nothing harmful about it.

Hi Justin,

Just a small advice about the type of membrane that you used. Usually nitrocellulose membranes are not recommended for stripping, PVDF are more recommended for that purpose.

Another things is as you loose some protein at each stripping steps so you should start with blotting proteins with the lower expression.

Washing steps after the stripping are also very important to allow a good bloting step.

Good luck for your experiment.

I use 0,2 M NaOH for 10 minutes for nitrocellulose. Before and after I wash membranes with water for 10 minutes to remove the tween and the NaOH respectively. It is very fast metod and it works good!!

Hello Mr Justin,

If you are going to strip ur blot 4-5 times, its better to use PVDF membrane instead of NCM.

PVDF has greater physical strength and can hold proteins to a longer extent than NCM.

Tris Glycine SDS at pH 2 works well. If you are distinguishing between low and high MW proteins, then it is also possible to cut the gel in half and strip each half fewer times then the issue with mercaptoethanol being too stringent is alleviated. Finally, depensing on the source of your pimary and hence secondary antibody - e.g. if they differ between repeated incubations, you can simply quench the first reaction using e.g. 15% hydrogen peroxide. So if your primary incubarion was mouse anti A and your secondary is rabbit anti B, you can quench between these two and then perhaps strip the membrane before the third incubation, if the antibody is rabbit or mouse again.

In our lab we have used a commercial stripping buffer that is gentle and works very well with Nitrocelulose (the one that comes in the life technologies iBlot stacks at least). Its from GEBA http://www.geba.org/acojavascript:ntent%20catalogue/c106/1288.php sold in France by EUROMEDEX (No conflit of interests to declare:). I think it is not too expensive ~35eur per 500mL, just have to use enough to cover your membrane

Stripping in my lab is "no, no" because of the reason you've just stated - reducing the abundance of proteins and getting the signals of much worse quality. Why would you need to strip the membrane if you can choose the loading control of other MW? I would prefer to pay 13$ for extra gel, but get nice clean bands without doubts.

@Edita: You do bring up a good point about stripping reducing the overall quality of the blots in general, and if it is simply the extra labor of pouring a new gel and doing the transfer that is in question, I would immediately agree with you that it's definitely worth it to just do more gels.

However, what if samples are very precious and hard to get? For example, I am doing blots of FACS isolated cell populations which yield perhaps 80k cells from 10 transgenic mice. I can barely get enough protein to run one gel using conventional blotting techniques. Do you guys use some kind of tertiary detection method other than the standard biotin-secondary, SA-HRP->fluor rxn that allows much lower protein loading?

In our lab we routenly use the re-stripping buffer for re probing the transferred protein on NCP membrane. The concentration of the reagents employed in the buffer as follows. 62.5mM Tris -HCl (pH 6.7),100mM beta mercaptoethanol and 2% SDS . Keep the NCP membrane in this buffer at 56 degree celscius for 1h followed by extensive washing using PBST and PBS. Ensure that the smell of the beta mercapt is almost nil . At this time point the blot is ready to be kept for blocking and for overnight 4 degree celsius and then u can re probe this blot with ur antibody of interest.

STRIPPING BUFFER (2% SDS, 62.5mM TRIS pH6.8, 100mM Beta-mercaptoethanol (bME)

Reagent 500ml

20% SDS 50ml

1M TRIS pH6.8 31.25ml

H2O to 500ml

ADD THE bME FRESH BEFORE USE*

Hi u can look into the abcam site for the protocol of a mild stripping buffer,,,...it has worked well for me

There is a really good protocol on abcam, it works well. Link: http://www.abcam.com/ps/pdf/protocols/stripping%20for%20reprobing.pdf