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ICSVortex 10-11-2012 07:47 PM

western blot bands too strong
Hi guys,

I am attempting a western blot, and probing for beta-tubulin as a loading control. I loaded 40ug of total cell extract per lane, and the bands for my protein of interest look great. Unfortunately, when probing for beta-tubulin the bands come out too strong. I am exposing it to x-ray film and it is becoming saturated even for a 1 second exposure (I can see my bands glowing on the membrane in the dark room). I diluted my primary and secondary antibodies 1:2500, and I suppose I can go to 1:5000 if necessary. Any tips would be appreciated. Also, can I simply wait for the HRP to use up more of the substrate to get weaker bands? I was thinking that stronger bands might use it up faster and appear weaker than those that are not as strong to begin with (if that makes any sense), which would be a problem. And how long does it generally take to use all of the substrate (minutes? hours?). Thanks!


Kneeanderthal 10-22-2012 06:17 PM

Re: western blot bands too strong
Load less (maybe 20 ug) when probing for beta-tubulin and dilute HRP antibody 1:5000, even 1:10000 (had the same problem with an anti-strep HRP). You could wait but I've seen the HRP active for days even after removing the substrate.

Adz85 11-20-2012 09:57 AM

Re: western blot bands too strong
We dilute up to 1:25000 secondary antibody when we do beta-actin. You could also try diluting your ECL detection reagents. We've found Milipore's to be much stronger than Amersham's and needs to be diluted more when detecting our loading control proteins.

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