ELISA - subtyping; rtPCR analysis - plus the chromatographic purification, Western and IHC you've already done will be ample.

Stick with BSA/PBS. You can also use the Jackson Immunoresearch normal gt or rb Ig, or any other low fl prtn. Just change your plate and step (5) of the protocol above. Given your charged peptide, its probably binding directly to the plate (and 'high binding' plates are charged) Milk solids emit ...

Don't try to use block such as Blotto or casein in your assay. Try using uncoated/uncharged/untreated polystyrene plates (all black) for your assay. Do the peptide binding at ~8dC (refrigerator) for ~ 1 h.

You raise a very good point - I always get my techs or undergrad to do a titration when they first do these assays, since buffer, # cells, plate surface, flat v/s round bottom are all variables that need to be addressed. I've found that a range of 0.5-2 ug/ml suffices for a strong signal (some of my ...

Hi! For plate bound Abs, your coating conc is your 'final' concentration (accepted policy, even though not all your Ab may bind). Thus, for 2 ug/ml in a 6 well plate, I'd recommend 2 ug aCD3 in 1 ml of buffer (see previous post), o/n coat at ~8dC (refrigerator). Your culture will probably be done in ...

One quick suggestion is that you might want to do the incubation in a refrigerator (~8dC) for ~30-40 mins, followed by 2x wash with cold HBSS+1%BSA/0.5% FBS. (Do not incubate on ice (0-4dC) for this app). You can use the wash buffer as the Ab incubation buffer too. What colors are you using on the p ...

There are several methods - and I usually prefer multiple readouts in cell death studies. For macs and T cells, a mix of the following works well from a publication standpoint. IF: DAPI (total)+BrdU(TUNEL: DNA frag)+FLICA (active caspase) FACS: Annexin/PI, cell cycle PI, FLICA, JC1 mitochondrial ...

If you have lots of conditions in a plate and want to assay 'untouched' cells, I'll recommend a combination of active-Casp3 (cells on plate - e.g. Promega Apo1 casp assay) + lactate dehydrogenase assay (from sup, to detect lysed cells; many vendors). I've used Mediatech's cell dissociation buffer ( ...

I've used Bradford at 1:250 (microplate reader) using a similar buffer to yours, so given both pippuri and my experience with it, there is no reason for you to stop your work. However, BCA does work better across a range of sample concentrations and compositions - I recommend the P ...

I think the Alamar Blue format is supposed to be a more stabilized form of the resazurin salts that are a part of some microbiological culture media. Thats what enables readings even after you take it out of a CO2 incubator. All the best for your assays! -sam