5 replies to this topic

[TerrorTabby]

Is is possible to strip a blot too much? I recently stripped a blot of mine and reprobed for beta-actin. While I got a signal for beta-actin, the bands were very funny looking. They were incomplete bands and it looked like only half of the band was giving a signal. The bands for the initiate protein I probed for looked normal. Perhaps, I did not wash off the stripping buffer well enough but I thought I washed the blot thoroughly. The protocol I followed was the following:

- Incubate membrane in stripping buffer for 45min at 50 degress
- Rinse under tap water for 1 hr
- Wash in TBST x2 for 10min

Stripping Buffer
10ml 10%SDS
6.25ml 0.5M Tris-Hcl pH 6.8
0.4ml beta-mercaptoethanol
33.57ml ddH20

Edited by TerrorTabby, 12 June 2011 - 02:01 PM.

[casandra]

TerrorTabby, on 12 June 2011 - 02:01 PM, said:

Is is possible to strip a blot too much? I recently stripped a blot of mine and reprobed for beta-actin. While I got a signal for beta-actin, the bands were very funny looking. They were incomplete bands and it looked like only half of the band was giving a signal. The bands for the initiate protein I probed for looked normal. Perhaps, I did not wash off the stripping buffer well enough but I thought I washed the blot thoroughly. The protocol I followed was the following:

- Incubate membrane in stripping buffer for 45min at 50 degress
- Rinse under tap water for 1 hr
- Wash in TBST x2 for 10min

Stripping Buffer
10ml 10%SDS
6.25ml 0.5M Tris-Hcl pH 6.8
0.4ml beta-mercaptoethanol
33.57ml ddH20

If you could still see your initial bands, then I don't think you over-stripped. But your blots could use a longer wash too (1 to 2 hours)after stripping. Also, if you incubate the b-actin Ab O/N, perhaps you'd get a better signal. And if you really worry about your stripping buffer being too harsh, then you can try the mild one from Abcam protocol here...it usually works for most of our blots...

Edited by casandra, 12 June 2011 - 02:29 PM.

[steveneam]

Just curious, but what do you mean by 'over-stripping' or 'stripping'. Not really familiar with the terms. Thank you.

[TerrorTabby]

casandra, on 12 June 2011 - 02:17 PM, said:

If you could still see your initial bands, then I don't think you over-stripped. But your blots could use a longer wash too (1 to 2 hours)after stripping. Also, if you incubate the b-actin Ab O/N, perhaps you'd get a better signal. And if you really worry about your stripping buffer being too harsh, then you can try the mild one from Abcam protocol here...it usually works for most of our blots...

I incubated O/N with beta-actin at 4 degrees. The bands from the first protein were gone; I did not see them when I re-probed. My first post was not clear, sorry. When you say a longer wash (1-2 hours), you mean in TBST and not the tap water step, because I rinsed in tap water for 1 hour. Thanks for the suggestion of using mild stripping, I think I'll try that. Funny thing, I've done this harsh stripping before and I worked fine but now all of a sudden it's giving me problems. But isn't that always how it goes...

steveneam, on 12 June 2011 - 05:12 PM, said:

Just curious, but what do you mean by 'over-stripping' or 'stripping'. Not really familiar with the terms. Thank you.

Stripping means removal of the primary and secondary antibodies from the blot. I'm using beta-actin as my loading control so after I probed for my protein of interest, I strip the blot and re-probe for beta-actin to make sure I loaded all the lanes equally. I don't know if over-stripping is a real term. I've never heard anyone say it before. I made it up. I guess a better way to describe my problem is to say, "Did I strip the blot too harsh or not wash well enough?"

Edited by TerrorTabby, 12 June 2011 - 07:07 PM.

[protolder]

Hola when the proteins are separated enough from actin you can make this second WB withouth stripping. But if you need to stryp , thik that the process quit antibodies, blocking agent and a bit ob your bands, in other ways if you hybridyze with the same ab after stripping, the bands are less intenses. Buena suerte

[mswine scientist]

I've had this problem before. I switched to using a milder stripping solution. It's just 0.5 M NaCl and 0.5 N Acetic Acid. Incubate at 50C for an hour in this, then wash 5 times in standard wash solution. Note that this may or may not actually remove all your primary antibody, so it's best to use a secondary antibody raised in a different species than your primary antibody to prevent unwanted bands from showing up after stripping.