I have purified a sample of anti-peptide antibodies, concentrated them using ammonium sulfate, and dialyzed them overnight with 4 buffer exchanges. Then I desalted them using the zeba spin columns. I tried to find the concentration using a bradford assay (using another IgG as a standard) but I kept on getting a negative number. I know I have protein in my sample because I ran them on an SDS gel and I could see a band at 50 & 25. I need to find the concentration before coupling my antibodies so is there another way to concentrate antibodies other than using ammonium sulfate?
Do you have another method of determining protein concentration? If there is protein there and it is not coming up in bradford then there might be something in your buffer incompatible with that test. We use a BCA assay which is compatible for a range of buffers. Could you determine concentration via nanodrop (less sensitive I know) or ELISA against a standard?
If you have dialysed all the Ammonium Sulphate out well (and you use very concentrated amounts for this protocol) then I don't see how that would interfere with determining concentration. You can also use a spin column on your purified sample to concentrate.
Ways to concentrate:
1. Use ultrafiltration devices.
2. Suck out the water burrying a dialysis bag with Ab solution in a heap of sugar. Then tie the bag to the much smaller volume and dialyze the sugar out against preferred buffer.
3. Dry out the water hanging the dialysis bag in the fridge then finish like in 2.
4. Use dry Sephadex G25 in bulk. Centrifuge and collect the free liquid. This trick may require some tinkering.
I agree, another method for determining the concentration would be better. If your antibodies are purified, nanodrop or spectrophotometer would be fastest to determine it. And then concentrate down with spin column.
For pure IgG the extinction coefficient at 280 nm with a 1 cm light path for a 1% IgG solution is ~ 13.4 (some variation between species but insignificant for IgG estimations. Therefor if you place your sample (undiluted) in a cuvette (square quarts glass cuvette) with a one cm light path (standard cuvette sold most frequently) and read with UV spectrophotometer set to read 280 nm and blanked to 0 with same buffer without IgG you can estimate concentration quickly and easily. The equation above translates to absorbance of your sample (undiluted) measured at A280nm with 1 cm light path equals a reading of 1.34 = 1 mg/ml. For calculation A280 of sample / 1.34 = IgG mg/ml. I use an ultrafiltration device to concentrate samples.
As mentioned by others, I also concentrate purified IgG fractions by centrifugation (50kDa cut-off). I normally quantify the protein preparations by measuring absorbance at 280nm.
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