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:
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