Stripping membranes for WB - To strip or not to strip... ? (Jul/14/2005 )

Hi fellows,

I've been told that some labs do not strip WB membranes if the species-of-origin for a 1st antibody changes between two consecutive immunoblots (for instance, if the 1st antibody in a first immunoblot was generated in mouse, and the next WB will be performed with a rabbit-generated 1st Ab).

I'd like to hear your opinions and experiences with this lots-of-time-saving method. Currently, I strip my PVDF Mb's before every single procedure...

Regards,

-Carbio-

QUOTE (Carbio @ Jul 14 2005, 04:21 PM)

i do not think the species matter. but stipping sometimes cause the loss of signal which makes it hard to interpret.

-pikip-

If you are a girl, strip.

If you are a guy, keep your clothes on.

Just a joke.. don't ban me.

-pBluescript-

QUOTE (pBluescript @ Jul 14 2005, 10:40 PM)

If you are a girl, strip. If you are a guy, keep your clothes on. Just a joke.. don't ban me.

Ha, ha, ha... good one. However, I don't see your comment or opinion about the topic, my friend. I bet you have something to say about it.

-Carbio-

hi
i always strip my membranes. I've tried to strip three times the same membrane and the results where similar (by chronometered exposure time and developing in a machine)... so i've keeped in mind that point...

-fred_33-

QUOTE

However, I don't see your comment or opinion about the topic, my friend. I bet you have something to say about it.

Actually, no. I've never used PVDF for western blots. So my opinion would be rather useless.

-pBluescript-

I have stripped my pvdf upto 4 times ...works fine.

Definitely strip
p..

-Pria-

I strip membranes using a SDS/mercaptoethanol buffer and my primary seems to stay on with some affinity. Incubation in Amersham's ECL plus for 40 minutes was required for the same signal that I saw before I stripped. So the primary was still there. Other NaOH buffers are harsh and tend to take the target protien off of the membrane. However, this may be required with some samples.

It appears to take some optimization and finesse.

-Randomancer-

Hi,

I am not a big fan of stripping, mostly because it takes time and it smells bad!

Here is a good reference about this topic: BioTechniques 28:216-218 (Feb 2000)

Here is a quote:
The major idea for this method (direct reprobing) is based on the theory that the binding sites of the target protein and its primary antibody are saturated, and horseradish peroxidase conjugated on secondary antibody has been inactivated totally by hydrogen peroxide in substrate during the first detection.

-Junon-

hey
hopefully you are using ECL methods.
or not using the fluorescent dye odyssey method, b/c i just wanna concentrate on talking about the shortcomings of this method

because if you use odysssey, chances are you won't be able to fully strip off the dyes. (here we use fluorescent 2nd Ab)

also, i have to switch from odyssey to ECL because some of the Ab's won't work very well with odyssey 2nd Ab's.

but if you actually can fully strip the dyes, then go ahead!!!

-cescfabregas-