I'm trying to isolate and measure the concentration (absorption at 280) of hemocyanin from spider blood, but I am only able to reasonably get around 20-30 microliters of starting biological sample. Packed chromatography size separation columns seem to require 100+ times more starting samples than I can produce. Does anyone have any suggestions on how I could isolate/purify from a sample as small as 30 microliters?
Hi Rachel,
If you want to keep your protein in native state you should use combination of narrow-bore gel filtration column Superdex 200 PC 3.2x30 from GE Healthcare and narrow-bore ion-exchange column (depends on isoelectric point of your protein) for two-step purification procedure. If native state is not significant try purification on reversed-phase HPLC column C4 with wide pores (300 A).
While high resolution microbore chromatography can be great, it can also be expensive if you don't have ready access and expertise. Is there an antibody to your protein target (hemocyanin?) that can be used to develop a radioimmunoassay? This seems much more sensitive and would allow high throughput, should that be desirable. In order to use A280 to quantify you will need to get your protein quite pure, which seems very daunting for such small amounts of starting material.
I'm inclined to agree with Richard. What is the starting concentration of your target in the blood vs. other proteins. If it's really high, you might have a chance with ultra-small scale physical characteristics as Sergey suggests. But if it's low, this is going to be too difficult, and you'll have to do what Richard says. (have an antibody made based on peptide sequence, if you must!)
With blood glycoproteins in particular, a lot of the physical characteristics (charge, hydrophobicity, size...) overlap, because the column will "see" the characteristics of the sugars, which aren't going to be different enough to separate one protein from a large excess of others.
Also, if this is a hemocyanin, don't you have a better wavelength, where you can see the heme? It should be far in the visible range, where all other proteins are dark.
I have different treatment groups, such that the concentration should be quite high in some, but quite low in others. I'm guessing that my best route at this point is to have Abs made, and try to get them out that way. Hemocyanins can be read around 278nm, but I suspect that there will be other proteins in that range. So I would still need to purify to get an accurate measurement, correct? Or is there a more crude way?
Please find the attachment for selection of right method. I would recommand Ion Exchnage chromatography for this as pI 4.5 - 5.5 and also have isomers of hemocynin.
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