Hana: Optimal concentrations for proteins adhering to plastic wells varied depending on the size, hydrophobicity, purity, etc. of the protein of interests. One should also be aware of the fact that the plasticwares used to dilution can also remove/bind your protein. Therefore, it is an empirical issue to be answered in practice, though there is a maximal binding capacity of the wells specified by the manufacturers. Good luck.
Hi Hanan do you mean what sample dilutions should you use on the ELISA?
If so then you will have to determine this empirically, The kit you have will give a working range depending on the kit's sensitivity (i.e. 500 pg - 5 ng).
Elisa kits generally have 96 wells in strips of 8 (12 x8 strips).
I would suggest (if you have sufficient spare wells and sample) running a test strip.
i.e. take one 8 well strip and run a few samples on it to determine the rough amount of IL-6 you have in you samples.
On this strip (following the kit protocol) run at least 4 points of the 8 point standard curve (S1, S4, S7 and S8). S8 can be "dilution buffer" or "media only". Generally you need to get signal above S7 for it to be in range of the kit.
With the remaining 4 wells of the strip run various dilutions of what you might expect to be the samples with the highest and lowest levels of IL-6 in them.
i.e. Well1 (neat), well 1 (1:10 dilution), Well 8 (neat) well 8 (dilution).
This should give you a rough idea of the dilutions you will need for the remaining samples (which you can run on the remaining unused strips). Elisa kits generally provide sufficient reagents (excess) to generate two sets of standard curves etc.
I have had to do this a number of times for a lot of different Elisas and whilst it sometimes is not informative (i.e. when the kit is too sensitive and I have a very high level of protein in my samples) it is still better than going "blindly" into an Elisa.
Hope this helps,
Gary
If you are using cell culture media, then as low as 50 microL media works. For tissue, I've used as low as 1 microG protein and it gives me a signal. When using serum, I take 200ul serum and dilute it 5X for ELISA. Although, this all depends on your conditions or the type of tissue you are using. R&D systems ELISAs are pretty sensitive.
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