about western blot - Please help me (Oct/22/2006 )

Hi. I am new in western blot. My secondary Ab is a HRP one. Do you think If I want to do the bands densitometery, I must change it to an ECL type one?

Another question: what is sample buffer? I see in some papers that they lyse their cells by sample buffer. Do you think it is better than RIPA buffer?

Thanks

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Hi,
HRP is a type of secondary antibody which is developed with the substrate ECL, and you will obtain bands to densitometer. If you have bought an HRP secondary antibody, then you will have to buy an ECL substrate to develop the bands.

For me, sample buffer is "Laemmli buffer", which turns blue when you mix it when the sample and allows you to see the front migrating on the gel during the electrophoresis. RIPA buffer I think it´s a lysis buffer, not a sample buffer like Laemmli.

hope this helps although i´m not an expert

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Hi pumuki, thanks.
and how we can do the densitometery of the x-ray film? I mean how we can convert it to a file to analyse?
thanks

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If you don't have an image acquisition equipment, you could scan the film and save as a .TIFF file. It works for the densitometric analysis with any software. If you don't have any you can download the ImageJ software from NIH website, it's free. This program can analyse files even in .JPG format. Other programs are Quantity One, Prism, etc.

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Yes, for example I have a densitometer from Biorad, which is an scanner with very good resolution. But with a regular scanner you can do it, obtain the file and analyze the bands as aleruiz said.

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Yes, we have some image analysers. Thanks a lot.

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if not using ECL, you may stain classically direct on the blot, and soak the blot in DMSO (protocol of Stein et al.; (?) forgotten the year...) so that it gets transparent for densitometry

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[/quote]

if not using ECL, you may stain classically direct on the blot, and soak the blot in DMSO (protocol of Stein et al.; (?) forgotten the year...) so that it gets transparent for densitometry
[/quote]

And how I can convert the blot to a file?

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[quote name='saya' date='Oct 24 2006, 03:34 PM' post='74305']
[/quote]

if not using ECL, you may stain classically direct on the blot, and soak the blot in DMSO (protocol of Stein et al.; (?) forgotten the year...) so that it gets transparent for densitometry
[/quote]

And how I can convert the blot to a file?
[/quote]

the same as with ECL blots, with scanner for transpirancies;
if there is not possibility for transparent/positive film scanning, you may scan without transparent light but then there is no need for making the blot transparent; transparent/film scanning give much better results in densitometry

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Very complete. thanks Kosmodrom.

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