Hi there,

Of course you can use a water bath! What I usually do for small size membrane is to roll it so that it can be inserted into a 50mL Facon tube filled it with the stripping solution.

A waterbath with a lid worked fine for us, we put the container with the membrane like a little boat directly into the water. Good luck for the stripping! For me the biggest obstacle is to have an even removal of the antibody and that the stripping does not bias my result. Instead of stripping we often cut the membrane into stripes and can probe for the different targets in one overnight incubation. Clearly this does not work if your targets of interest travel at the same kD.

Alternatively, you could try 10mM NaOH (higer concentrations will dissolve nitrocellulose, if nothing else works, switch to PVDF) or a buffer with pH 2..3 (e.g. Glycine-HCl) at room temperature.

Hi Chichi Okagbue

I have carried out stripping on nitrocellulose many times with the following recipe:

For 200 ul stripping buffer, add 3 g Glycine, 200 mg SDS and 2 ml Tween. Then adjust the PH to 2.2 by HCl. This recipe works in RT and it is very effective.

Best ....

Has it been tried previously?

Dominique Liger Thank you for your reply! Would you recommend the falcon tube method for a membrane containing a 15 well gel?

Anika Stracke Thank you for your reply! I will try the water bath method and hope for the best

Wolfgang Schechinger Thank you for your reply! I've stumbled across the low pH glycine method a few times so I will probably try this out and compare to the heated BME method.

Mohamed Khashan Thank you for your reply! Roughly how long do you leave the glycine buffer on the membrane and also how long do you wash off the buffer for please. Many thanks

Ali Ahmed Alawady yes it has been tried previously but since we have since moved buildings so trying to find an alternative I can use to incubate the membrane for 50 degrees

Hi Chichi,

We wash with it two times 10 minutes each, then wash 3 times with PBS 10 min each after that wish TBS-T for one time 5 min. Subsequently, go ahead with blocking and other steps.

Best...

Mohamed Khashan Thank you so much for this!

Hi again, just to answer your specific question:

If the transfer is made from from a minigel, the membrane should be around 9cm x 6cm which fits very well into a 50mL Falcon tube.