First time ELIZA? protocol? - (Sep/30/2009 )

Hi,
I want to do ELIZA for the first time. Can any one help me with the protocol. I have primery abs. No one here can help me with how to do it. Which secondary I should use? Which substrate I need to use to get colour?
can you please kindly help me?

Thanks,
Sara

-saraarasus-

Please give us some details so we can help. What is the analyte you are measuring? What is the specificity and specie and purity of the antibodies you have? What is the enzyme you wish to use? Is your secondary ab conjugated?

-sgt4boston-

Hi,
I am measuring the apoptosis family proteins. I want to check their expression level in cell lysat. I have secondary ab which is HRP conjugate.
Please tell me if you need moreinformation.

Thanks

sgt4boston on Oct 1 2009, 02:55 AM said:

-saraarasus-

Hi,

Thanks for your the website. I was looking for the protocol that you send it. I will try it and ask you Questions accordingly.

Cheers and thanks,
Sara

CellSpecific.com on Oct 1 2009, 10:37 PM said:

It sounds like you're just asking what secondary antibody will be able to detect the primary antibodies you already have. Yet, it sounds like you already have a secondary antibody? Anyway, provide these information and we'll be able to help:

1. From sgt4boston: Tell us the species origin of your primary antibodies (e.g., rat, mouse, rabbit)
2. Do you know the isotype(s) of the primary antibodies you have (e.g., IgG, IgG1, IgG2a, IgG2b, IgM . . . )

I like to use TMB substrate with HRP (e.g., http://www.piercenet.com/Products/Browse.cfm?fldID=01041009)
Here is a typical ELISA protocol from abcam (http://www.abcam.com/index.html?pageconfig=resource&rid=11422) to get you started with coating/blocking/incubation with antibodies/developing and more . . .
Here is a nice illustration of step by step sandwich ELISA of the type you'll likely be running (http://www.raybiotech.com/elisa.asp)

Hope this is helpful.

-saraarasus-

Don't forget that you also need protein to run for a standard curve.

-tlp-