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Western blot membrane imaging


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7 replies to this topic

#1 junbug

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Posted 17 October 2012 - 02:05 PM

Hi,

for the imaging, do i have to use either x-ray film or digital imaging station?
is there any other way to do it?
Thanks you

#2 Curtis

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Posted 17 October 2012 - 08:32 PM

it depends how you develop your blot. for example if you use AP-conjugated antibody and develop using BCIP and NBT then you can use a normal camera, but if you develop with ECL solution (which is very popular) you may need chemiluminescent gel doc or system. I used to develop with X-ray method, but I don't recommend it, because the reagent you use stain everywhere.

#3 bob1

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Posted 18 October 2012 - 12:05 AM

There are also fluorescent tagged antibodies, which are often in the IR range for fluorescence and have incredibly stable signal. The equipment for using them is quite expensive though.

Film is still the standard for these sorts of things, most of the cheaper camera based systems, such as the Biorad ones are terrible for taking images of chemiuminescent blots.

#4 junbug

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Posted 18 October 2012 - 06:48 AM

it depends how you develop your blot. for example if you use AP-conjugated antibody and develop using BCIP and NBT then you can use a normal camera, but if you develop with ECL solution (which is very popular) you may need chemiluminescent gel doc or system. I used to develop with X-ray method, but I don't recommend it, because the reagent you use stain everywhere.

Do you mean I can even take a picture of it with my phone camera in shiny room if I develop using BCIP and NBT?
I've using Kodak imaging station in another lab, but current lab doesn't have that.
We have Polaroid mp-4 land camera and ultra-violet light. Some one in our building must used this before because I found biomax film. But nobody knows who used that.
So I'm wondering if this camera would work with chemiluminescent.
Any advise is appreciated.
Thanks

#5 Curtis

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Posted 18 October 2012 - 09:12 AM

Yes,but you can only stain once.I mean you can't strip the membrane and lable with another antibody. but since you can actually see the bands with naked eye it is still my prefered method.

#6 junbug

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Posted 18 October 2012 - 10:00 AM

So I guess I can take the picture of the membrane in dark room with a traditional film with camera if I use HRP and ECL.
Is this correct?
Thanks

#7 bob1

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Posted 18 October 2012 - 02:27 PM

Correct, you can use a darkroom and a camera, but you need a camera capable of long exposures and low f stop (preferably f1.4 or higher) as the amount of light put out by a typical membrane is not very high - even x-ray film often requires exposures of 20 min or more.

#8 junbug

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Posted 19 October 2012 - 06:08 AM

Thank you people




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