stripping with acetic? - (Sep/17/2006 )

hi,
I was told to do the stripping following this protocol: wash 15 min with acetic 5% and then wash 5 min x 6 times with TBSTween, but it didn´t work.
I had never heard about stripping with acetic, should I try other protocols with glycine, B-mercapto, NaOH? Any premade solution works well? (I don´t have a fumehood for the b-mercapto).
thanks

---

we use a strip solution from Pierce, it is ok; glycin pH2 protocols or massive SDS with mercaptoethanol or 4M guanidine thiocyanate also work; but I must admit that I have never met the ultimate stripping solutions...so if anybody has THEE stripping buffer to recommend - let us know...

---

I am actually trying to find that right now too. I can tell you what doesn't work:

2%SDS, 100mM Bme, 62.5mM Tris-HCl pH = 6.8
*Tried at 65C for 1.5-2 hrs
->I didn't get anything on reprobing with four different antibodies (they were blank, which means that maybe the protein was also stripped off?) on four different blots stripped this way but haven't yet investigated further
*Tried at 50C for 1 hr
->Didn't strip all the way

Pierce stripping buffer
*Tried at RT 30 min, 1 hr, also tried at 37C for 1 hr-> none stripped completely

pH = 2.5, 0.2M Glycine with 0.05% Tween20
*Tried at RT for 30 min -> didn't strip completely

I am going to try the pH = 2.5 buffer next at 80C for 20 min because I saw that someone on this forum posted that before. I have used the guanidine buffer you mentioned above in a different lab, and I got really high background after reprobing.

---

I used to strip with this method for several years now.
I do it for 30 minutes only, with shaking.
wash extensively and re-block.
I'm able to strip and re-blot twice. sometime I still have some slight signal from the antibody that should have been stripped, but only when I expose the film one hour, while I got the signal in less than 1 minute before stripping. (that's why I prefer to always blot first with the antibody that gives the lowest signal).

I tried Pierce stripping buffer. the first bottle I bought was perfect. I had a better signal after stripping with Pierce buffer than my SDS-BME buffer. However, the second and third bottle were not able to strip as well, and I was using the same antibodies. I came back to the SDS-BME buffe.

---

Thanks everybody.
I´ve found that Chemicon and Calbiochem also sell a stripping solution. Has anybody used them?

---

Missele--

What temp do you use the Bme/SDS buffer at?

Thanks!

---

50°C, as you did.

did you try several antibodies? Maybe you have one very resistant !

---

no, at the moment I´ve only tried to strip one antibody. I will try with another to see what happens.

---

Hi Missele,

I guess I am just wanting the "perfect" stripping buffer that will always work for everything. But I guess that's not going to happen! Thank you!

---