I use MHC Class II tetramers alot in my studies. I'm wondering if someone has used fluorochromes OTHER than APC, PE for tetramers.If so, how did you go about making the calculations for conjugates, ie getting the molecular weights of Streptavidin-Fluorochrome conjugates?
We do this on a regular basis for PE and APC tetramers using the description found in the NIH tetramer core (http://tetramer.yerkes.emory.edu/client/faq#1). They have calculated the MW of Strepavidin-APC, PE and FITC conjugates. I'd like to have my tetramers on an alternate channel, I was thinking that the new Brilliant Violet dyes from Biolegend/BD would be a good bet. Biolegend gives the conjugate MW (http://www.biolegend.com/streptavidin_conjugates). However, looking at that list, their reported MW for APC is 260KDa. At the NIH core, the reported value is 160KDa. So I don't know if it's possible to use the values that Biolegend reports.
Hi
I have used streptavidin conjugated with PE/Cy7 from biolegend to make MHC class I tetramers. It worked perfectly fine. When I talked to their supplier they told me that the concentration mentioned is only for the streptavidin content alone (not streptavidin conjugate) which mean I used only the Mw of streptavidin for calculations. Check this with the supplier. Otherwise the Mw can of cause differ depending on how many flurochromes is attached to the streptavidin this is not always 1:1 ratio...
Good luck
Rebecca Axelsson Robertson, PhD
Thank you! So you assumed the concentration (0.5mg/ml, 0.1mg/ml) was based on streptavidin MW alone and it worked?
Hi, I would like to add a critical point about the source of streptavidin as your question is specifically about class II tetramers. Along with NIH tetramer core, we did a head to head comparision of streptavidin conjugated with APC or PE from three different companies (Invitrogen, Leinco and Prozyme). We generally use Invitrogen streptavidin for class I tetramers but for class II tetramers conjugation with streptavidin from prozyme gave a superior quality stain with less background. We haven't tested the one from Biolegend, hope it works for you. Unfortunately, Prozyme doesn't have fluorochromes other than APC and PE.
Ata
Thank you Ata, I checked out their website. I'm going to guess that the improved sensitivity is due to their SAv conjugates making de-facto octamers (since they described having 2-streptavidin molecules per PE molecule in one reagent, determined by HPLC). It would be nice to get more sensitive reagents...but my PI would never go for it, because he wouldn't see the value of buying new reagents (unfortunately for me! It drives me crazy to get so much background noise sometimes). The SAv we have for PE and APC is eBioscience, do you know how that stacks up to prozyme? How did the streptavidin conjugates from the other companies stack up as well?
As mentioned earlier, I did not have a chance to compare eBio streptavidin with other sources but you can check with Rick Wills at tetramer core.
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