dual elispot

[futuremsfdoc]

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anyone here ever run across ap (alkaline phosphatase) conjugated antibodies against fitc, il-10, or ifngamma?

 

[futuremsfdoc]

these companies that have them only sell them as part of a kit and i just need the antibody.

 

[ImmunoANT]

lots companies sell these kinds of Ab. Jackson, Santa Cruz (be warned), Cell Signaling came to mind. BD Pharmingen is my favorite but they are expensive. You can also go to Biocompare - they let you do price comparison.

 

[ImmunoANT]

BTW, IL-10, IFN-g? are you testing Th1/Th2 polarization? 🙄

 

[Treg]

i have a friend who is a rep for ebioscience, and they have great, inexpensive antibodies. You should check them out 🙂

Treg

 

[psyuk]

hi Treg, is your Foxp3 expression level normal?

😀

anyhow i agree with immunoANT that you should be careful of what you buy from santa cruz.

 

[randallB]

check upstate

 

[futuremsfdoc]

BTW, IL-10, IFN-g? are you testing Th1/Th2 polarization? 🙄

looking at whether or not regulatory cell subsets implicated in suppressing diabetes development may produce both il10 and ifng.

btw, no luck on the antibodies so far. i already have anti-il10 and anti-ifng biotinylated, so i was thinking if i use anti-il10 biotin and anti-ifngamma with fitc, then use an anti-fitc ab with hrp conjugate i would be able to develop the dual elispot. no one wants to sell me the anti-fitc hrp conjugate separately though. damn those greedy biotech companies!

 

[microTAS]

for fitc, I recommend Jackson:

http://www.jacksonimmuno.com/2001site/home/catalog/antifitc.asp

 

[microTAS]

Since we're on this topic, I have two additional questions:

1) What assay buffer formations do you guys use? E.g. % BSA, Tween/Triton, Tris or PBS

2) Anyone who's worked w/ magnetic bead sandwich immunoassays have any tips?

Also if you know a place where I can looked this up, that would be really helpful!

 

[ImmunoANT]

1) What assay buffer formations do you guys use? E.g. % BSA, Tween/Triton, Tris or PBS

for what kind of experiment? with tween or/and triton, these are detergen, so very likely you either want to lyse the cells (0.8-1% should be sufficient) for WB or ELISA or want to permeablise the cells for intracellular staining. For intracellular staining, i usually use BSA, triton, saponin with 1X PBS. can't remember the exact recipe (have to check my notebook but am not in the lab right now)

2) Anyone who's worked w/ magnetic bead sandwich immunoassays have any tips?

again, you probably have to be a bit more specific. But when it comes to applications with magnetic beads, miltenylni is the best.

ImmunoANT

 

[ImmunoANT]

btw, no luck on the antibodies so far. i already have anti-il10 and anti-ifng biotinylated, so i was thinking if i use anti-il10 biotin and anti-ifngamma with fitc, then use an anti-fitc ab with hrp conjugate i would be able to develop the dual elispot. no one wants to sell me the anti-fitc hrp conjugate separately though. damn those greedy biotech companies!

you may also want to check what kind of host the Ab is raised from because you don't want to have cross-reactivity. Also with that, if you can't find HRP-conj anti-FITC Ab, maybe you can try to find a HRP-conj Ab that recognizes the specific isotype of the anti-IFNg Ab. For example, if that anti-IFNg Ab is raised from goat, then you can detect that anti-IFNg Ab (whether it's conjugated to FITC or not) with Anti-goat IgG Ab conjugated to HRP.

hope that helps.... good luck

whew..... don't you just love immunology *sarcasm*.....

ImmunoANT

 

[flyingillini]

Horseradish peroxidase!!! 😱

 

[microTAS]

Hey ImmunoANT (or anyone else),

What vendor do you get your BSA from? Right now, all I have is a low grade from Sigma

Thanks!

 

[ImmunoANT]

Hey ImmunoANT (or anyone else),

What vendor do you get your BSA from? Right now, all I have is a low grade from Sigma

Thanks!

depending on your purpose. if it's for ELISA, then you want BSA without fatty acid and globulin (therefore, more expensive). if you want BSA just to serve as buffer for molecular/cell bio procedure, then regular grade BSA should do fine. If you want to use it for cell culture, remember to pick one that's low endotoxin (and of course sterilize/filter the solution beforehand) Sigma Aldrich sells all these kinds of BSA. When I started at NIAID, I bought a bucket of molecular bio grade BSA (~ 1Kg), and 2 years later, we still had more than half left... oh..... I miss the life in a lab. I'm...... well, how should I put it....... between labs?! 🙁 🙁 🙁

 

[microTAS]

Alright cool, thanks for the info. I ended up buying Jackson's BSA specifically for sandwich assays. Sigma's globulin free and globulin/FA free BSA was expensive!

Also, you definitely sound like a MSTP student 😀 👍

 

[ImmunoANT]

Also, you definitely sound like a MSTP student 😀 👍

don't know if I should take that as a compliment or what. 🙄 🙄

 

[futuremsfdoc]

thanks for the advice all. the jackson abs will be perfect.

 

[microTAS]

don't know if I should take that as a compliment or what. 🙄 🙄

Ha, it's a compliment! Nothing wrong w/ enjoying your work