4 replies to this topic

[sc_queen]

I just wanted to be sure I understand this correctly. So, if your primary antibody is anti-human IgG1, raised in mouse (mouse monoclonal), and APC-conjugated, then you would want to use anti-human IgG secondary antibody conjugated to the same fluorochrome (APC), correct? Then does this mean we need a negative control for every flurochrome we will be using?

e.g.
sample 1: anti-human mouse monoclonal (IgG2) CD49f-APC
neg control: anti-human mouse monoclonal IgG-APC

sample 2: anti-human mouse monoclonal (IgG1) CD34-biotin (with SA-PE secondary Ab)
neg. control: anti-human mouse monoclonal IgG-PE

sample 3: anti-human mouse monoclonal (IgG2a) CD24 (with anti-mouse secondary Ab-FITC)
neg. control: anti-human mouse monoclonal IgG-FITC

Correct?

Also, how do you choose a company when you buy your IgG control? It probably won't matter too much, but I am just curious.

Thanks!

Edited by sc_queen, 27 January 2012 - 02:41 PM.

[Tabaluga]

You did understand it correctly. Your control should be conjugated to the same fluorochrome as your sample. Note that you should use the same isotype in sample and control, e.g. when you have mouse IgG2a for your sample, also use a mouse IgG2a isotype control.

[scolix]

yep, agree with the above post, you need to have the same isotype and subtype,

[sc_queen]

Thanks! It helps.

[keefec]

And I believe you just need one sample control with all 3 IgG control antibodies in it. unless they crosslink.