I am not quite sure of what exactly you are asking. If you are asking for ways to develop your blots, there are many ways and also depends on the secondary antibody you use. If it is AP or HRP conjugated, chemiluminescence is the best way to detect your bands and perform volume analysis. There are also colorimetric assays available where you can develop bands on the membrane.

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2032.pdf

This should give you more details.

I could not understand the problem either. Do you mean transfer buffer? Then what do you mean by the blot analysis? Itr think Pooja raelly provided the best answer.

Hi Arvind,

I don't understand what exact problem you are having and what you have really asked for, but I do understand that you are giving problems to the contributors to understand and answer your question.

You are advised to formulate a clear question to expect a clear and defined answer from the fellow researchers. At the time being it seems to me that you probably want to know the compositions of the transfer buffer used in the WB analysis. If my guess is right then finding the answer is not at all difficult.

Transfer buffer (Semi-dry)

48 mM Tris, 39 mM glycine, 20% methanol, 0.04% SDS

A standard buffer for wet transfer is the 1X Tris-glycine buffer (25 mM Tris, 192 mM glycine, 10% methanol). For proteins larger than 80 kD, it is recommended that SDS is included at a final concentration of 0.1%.

To begin with look at the links:

http://www.abcam.com/ps/pdf/protocols/wb-beginner.pdf

http://www.cytographica.com/lab/solutions/transfer.htm

Best-

Question should be understandable to be get answered. Please ask question addressing your problem. Thanks

Are u are looking for fixer / developer or transfer buffer which is used for the transfer of blot from SDS-PAGE to Nitrocellulose/PVDV membrane?

Pooja Gupta-Saraf, Ru-Jeng Teng, Amritlal Mandal, Arvind Kumar,

Dear all:

I am trying to ask that, what is the transport media or what is the material that can be used for tissue storage for Western blot analysis.

Explanation:

In case if I want to submit the biopsied tissue to some diagnostic lab, which is far from my place. How do I submit the tissue, for histopathological techniques, we use formalin as a most common fixative agent/ transport media. But, In the present requirement of western blot analysis, the formalin usage was least recommended due to fixation of proteins. Hence, to submit the tissue for Western blot analysis, what transport media is required ?

Hope it is clear now, please let us know if the question is still obscure.

Hi Arvind: If it is tissue then you simply put it in liquid N2 or on dry ice to freeze it the keep in -80C. You can not use formalin since it will block the epitope for antibody to recognize and will not be able to run the western blot.

Dear Dr. Babu,

Dr. Teng already have given you the answer. Apart from using Liq N2 you can also consider following strategies if it seems feasible to you.

1. You can prepare the sample and transport them for WB analysis somewhere else or 2. You send tissue, as it is suspended in isotonic media/buffer.

For the sample prep you can use RIPA (1X, pH 7.4). Follow (Nitric oxide regulation of Na,K-ATPase activity in ocular ciliary epithelium involves Src family kinase, J Cell Physiol, 2013) paper in my RG page and for sample/tissue transportation as a second option you can use cold (2-4 deg C) isotonic Hanks Balanced Salt Solution (pH 7.4) (HBSS, calcium, magnesium, no phenol red, Life Technologies 1402509).

No way formalin could be used for this purpose.

Hope that helps.

Dear Babu,

We had kept the tissue in complete cell culuture medium for a week. The kept tissue cells were alive atleast for a week at 4 degree C . We checked viablity assay percentage of vialbe cells were quite good. Can you try this in your lab? See it works or not.

Thank you all, I will try with liquid nitrogen, since that is available in our lab.

I was sure about formalin use is not recommended, as it will block the proteins.