Hi. I measure my protein concentration using Nanodrop. and I get negative value. So, how can I proceed with SDS PAGE then? Thank you.
Hi,
one possibility is that something is wrong with your protein determination. When using nanodrop, this means UV-absorbtion? This is not very sensitive and can be disturbed easily. You should give some more information: How does your spectrum look like? What is the buffer you are using as blank and in your protein sample?
My recommendation would be to try another protein assay, Bradford or BCA to name my favourites. If you get nothing here: Concentrate your protein by precipitation (TCA/acetone) or ultrafiltration. Or simply run a part of your sample on a test gel and stain with Coomassie or silver.
Good Luck!
Hi,
one possibility is that something is wrong with your protein determination. When using nanodrop, this means UV-absorbtion? This is not very sensitive and can be disturbed easily. You should give some more information: How does your spectrum look like? What is the buffer you are using as blank and in your protein sample?
My recommendation would be to try another protein assay, Bradford or BCA to name my favourites. If you get nothing here: Concentrate your protein by precipitation (TCA/acetone) or ultrafiltration. Or simply run a part of your sample on a test gel and stain with Coomassie or silver.
Good Luck!
Hi there,
How does the Baseline look like? Once you have set the Baseline with buffer alone, immediately run it as a sample to see how it looks. It should normally be totally flat at zero absorbance on the whole window of wavelengths. If not redo the Baseline.
you can also just try running the gel and see if you can visualize your protein. If you have some protein of a known concentration, close to the size of your target protein, you can load several different amounts and use it to roughly determine the concentration of your protein by comparison of the staining intensity. Good Luck.
Did you check if your protein has any tryptophan, tyrosine, or phenylalanine residues?
Tryptophan is responsible for most of the absorbance of ultraviolet light (ca. 280 nm). Tyrosine, and phenylalanine absorb but not as much. Tryptophan is the rarest of amino acid. Most proteins contain ~1.3% tryptophan. So it is not hard to find a small protein that does not absorb UV well because it does not contain tryptophan... so you can't use this method to measure it.
SDS-PAGE stained with Coomassie (or silver stain if you don't have much protein) is the best way to find out which fraction contains you protein.
If the protein is not absorbing significantly at 280nm it will still absorb at 205nm because of the peptidic bond. So quantification may be performed at 205nm with diluted protein solution (5 to 50µg/mL) because specific absorption of protein is high at 205nm : 1mg/mL solution would give OD205nm=31. For more info on protocol and bibliography :
http://www.ruf.rice.edu/~bioslabs/methods/protein/abs205.html
Hello,
I agree with Mr. Nass, if Nanodrop results are negative, maybe you can try using different method for protein quantification. Try using BCA or Bradford method, and silver stain method for visualisation because it is more sensitive than Coomasie
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