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Re-blotting using a nitrocellulose membrane that was frozen

Re-blotting using a nitrocellulose membrane that was frozen - Western Blot Forum

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Old 08-23-2010, 11:18 PM
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Default Re-blotting using a nitrocellulose membrane that was frozen



Hi there,

I had some problems with my samples and I didn't get any protein bands. I know my antibody works because I ran a control and I got a band for that sample. So, I'm thinking something went wrong with my transfections. I saved my membrane blot and I frozed this at -35 C. I want to re-use this blot to try and detect a protein such as Actin or GAPDH (to see if my transfection was the issue). I'm unsure of what I should do when I take the blot out of the freezer. So my question is there necessary steps required for a blot that has been frozen? I was going to take the blot out of the freezer, let it thaw, wash it in wash buffer for 10 minutes (shaking) to remove any ECL reagents that may be present, THEN block it for an hour, and incubate it with my primary Ab and continue with my protocol as usual....Any suggestions? Has anyone used a blot that has been frozen.

Thanks for your help!
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Old 08-24-2010, 04:42 PM
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Default Re: Re-blotting using a nitrocellulose membrane that was frozen

I've re-probed dozens of frozen blots. What you plan on doing is exactly what I've done in the past - wash, re-block and probe.

If you've probed the blot previously you'll need to strip off the primary and secondary (I normally do that before I freeze), as they can survive freezing, leading to unwanted bands.

Bryan
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Old 08-26-2010, 02:55 AM
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Default Re: Re-blotting using a nitrocellulose membrane that was frozen

Thanks Bryan! I'm going to try this tomorrow!

Night.
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