Reg - western blot I have a doubt that I have cytochrome c primary antibody originated from goat polyclonal antibody and I don't have anti-goat secondary antibody, so shall I use rabbit secondary antibody means what will be the result? Either the band comes are not?
as per my experience band does not come. i tried same like you but not cytochrome c i used PGC1 A antibody
By "rabbit secondary antibody" do you mean "anti-rabbit secondary antibody"? This will not work. Switch your secondary antibody.
And do go and read a little on western blot theory... It will save you a lot of time and money.
You can start (but don't stop) here
http://en.wikipedia.org/wiki/Western_blot#Two_steps
You could use protein A with a horseradish peroxidase tag. Protein A binds to Fc portions, witohut discrimination of antibody specificity, or animal origin. Is your cytochrome c very diluted? usually you can see the red band on your SDS-PAGE or IEF while running.
Definitely you can use protein A with a horseradish peroxidase tag, but I fear much of the amplification effect will be lost. Also, will it actually be cheaper then commercial anti-rabbit-HRP Ab?
i am agree with yoram Gerchman , i have tried in my lab also surely there will be difference of amplication factor, so for better result you can go for anti-goat secondary antibody. Gud luck
U will never get a band on your western blot...... switch to anti goat secondary antibody,,,,, good luck
You have to use anti-goat secondary antibody if your primary ab is originated from goat.
Secondary antibodies are not as expensive as primary antibodies. You can buy good anti-goat IgG tagged with peroxydase to well known manufacturers like Sigma-Aldrich or R&D. It's better to use secondary antibodies to keep the aplification effect, especially if the concentration of your sample is weak.
You will most likely not get any bands if you try to use the rabbit, and even if you do, how could you determine what is real versus artifact? You need to purchase the appropriate secondary antibody. As Pierre-Marie says, they are very inexpensive and will ensure that you results come out correctly.
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology