Assistance with Western Blot

[zequeins]

Greetings all,

 

I plan to perform Western Blot on Human Hair Follicle Dermal Papilla Cells. But this is my first time performing Western Blot, I hardly know what to choose / prepare for the procedure. Specifically, I wanted to detect these proteins:

 

ERK 1/2

Phospho-ERK 1/2

Akt

Phospho-Akt

Bcl2

Beta-Catenin

FGF-7

 

As for the procedure, I am going to use RadioImmunoPrecipitation Assay buffer to lyse the cells. For the Antibodies, I've gotten a list of Antibodies for each of the proteins:

 

ERK 1/2: www.rndsystems.com/pdf/mab1576.pdf - Mouse Anti-Human ERK 1/2, 100 ug

Phospho-ERK 1/2: http://www.rndsystem...pdf/MAB1825.pdf - Mouse Anti-Human phospho-ERK 1/2, 100ug

Akt: http://www.rndsystem...pdf/MAB2055.pdf Mouse Anti-Human Akt, 100 ug

Phospho-Akt: http://www.rndsystem.../pdf/MAB887.pdf Mouse Anti-Human phospho-Akt, 100 ug

Bcl2: http://www.rndsystem.../pdf/mab827.pdf Mouse Anti-Human Bcl2 100 ug

Beta-Catenin: http://www.rndsystem...pdf/mab1329.pdf Mouse Anti-Human Beta-Catenin 50 ug

FGF-7: http://www.rndsystem...pdf/mab2511.pdf Mouse Anti-Human KGF/FGF-7 100 ug

Secondary Antibody: http://www.rndsystem.../pdf/haf007.pdf Goat Anti-Mouse IgG

 

I think my university should have the SDS-PAGE gel covered. I plan to perform the Western Blot on 4 groups of cells (1 control, 3 treated). However, since this is my first Western Blot procedure, I have a lot of questions regarding the issue:

 

 

1. Do you think the antibodies I have chosen are suitable enough for this protocol? When I do a search on the seller's website, there seems to be a lot of things to choose from / consider; how do I choose the right antibody for the procedures?

 

2. Do you think I should have chosen a different antibody for phospho-ERK 1/2 and phospho-Akt? Right now they are all produced from mice, should I choose a different source such as rabbit? If yes, that means I should get another secondary antibody, yes?

 

3. As you see, each antibody come in 100 ug size. How much do you think I will need for a single Western Blot procedure? (I will perform this 4 times, since I have 4 samples)

 

4. The secondary antibody I currently chose are HRP-conjugated; what else would I need to detect / quantify the Western Blot results using HRP-conjugated secondary antibodies?

 

 

I also welcome any suggestions you may give in this first attempt of mine. Thank you in advance for assisting me in this project.... and forgive my lack of knowledge in the matter

Edited July 3, 2012 by zequeins

[zequeins]

Thanks for helping me with this question guys. Should I / how do I delete this thread now?

 

(Help came in private message - you know who you are )

[microBloom]

I don't know much about Western blotting since I haven't used the technique, so please excuse this basic question, but what are the practical applications of using monoclonal vs. polyclonal antibodies?

[CharonY]

Monoclonal ones tend only to recognize a single epitope. For polyclonals are less specific in that regard.

[microBloom]

I read that monoclonal antibodies are better used for Western blots vs. polyclonal antibodies are used for immunoprecipitation, is this true? But if you were to use a polyclonal antibody for a Western, wouldn't those antibodies still only have to bind to the epitopes present on the protein of interest? Why would there be more unspecific binding?

 

Also, how would you determine if you should better use a mouse, rabbit, or other animal to get the antibodies from?

[CharonY]

A given protein can have a variety of epitopes. Polyclonals are essentially a mix of different ABs that may bind to different ones. Which one to use depends highly on what your assay is supposed to show. A monoclonal AB can, for instance be raised against a specific epitope, e.g. to identify proteins with a highly conserved structure (but which may be different in other respects). Conversely, you could raise a monoclonal that is highly specific for one particular protein only. In polyclonal you do not have that amount of control. Technically, if a polyclonal shows sufficient specificity you could use it in Western. However, you always risk that it will bind to other proteins, too. The unspecific binding is due to the fact that there may be IgGs that bind to epitopes that are not specific for that particular protein. But you generally would not know beforehand.

 

The animal to use depends mostly on cross-reactivity and tend to be more relevant for sandwich-type assays or for specific immunoprecipitation/depletion methods that rely on somewhat unspecific binding (which is used in some purification processes, e.g. to deplete high abundant proteins from a sample).

 

The source plays a relatively small role for the actual use of the antibody (it is more a production issue). However, one actual difference is found when using more distant organisms (e.g. chicken) which allows for a stronger reaction. That is, chicken IgYs can have higher avidty over mammalian IgGs, if raised against mammalian proteins (especially, highly conserved ones).