quantifications of western blots with imagej

#Quantifications of western blots with imagej series

Advantages of the Bradford Assayīradford protein quantification is quick, easy, stable for up to an hour, and isn’t affected by the presence of reducing agents in your buffer, like DTT or my favorite smelly lab chemical, β-mercaptoethanol.

#Quantifications of western blots with imagej series

In general, you measure the absorbance of a series of known concentrations of a standard protein to create a standard curve. Unlike the Folin-Lowry method, the Bradford assay doesn’t have a set endpoint, so you have to use this standard curve to calculate protein concentration based on its absorbance. It’s based on the interaction between Coomassie brilliant blue (you know, the stuff you stain your SDS-PAGE gels with) and the arginine and aromatic residues in your protein. When the dye binds to these residues, its maximum absorption shifts from 470 nm to 595 nm. There are good reasons that the paper first describing the Bradford Assay has been cited thousands of times! (2) The Bradford assay is an elegantly simple colorimetric assay for protein quantification. In which case, one of the following techniques might be more useful for you. This can be difficult to do especially if you freeze protein and use it for weeks/months but you no longer have the exact buffer to blank against. This doesn’t mean that UV-Vis is incompatible with DTT, it just means that you should use an exact DTT-containing buffer match to measure protein concentration more accurately. If you use DTT in your protein preps and use A 280 to measure protein concentration-be careful! DTT oxidizes over time leaving a product that also absorbs at 280 nm. Alcohols, certain buffer ions, and nucleic acids all absorb at 280 nm, thereby making this measurement non-specific for protein if any of these other molecules are present. To make matters worse, lots of other molecules interfere with this method of protein quantification. These factors alone make this approach unreliable. (1) Disadvantages of Measuring Protein Concentration using Absorbance at 280 nmĮvery protein has a different number of tyrosine and tryptophan residues and, annoyingly, you may not know the experimental extinction of your protein. If you’re working with purified protein samples and if you measure the complete UV-vis spectrum of your protein sample rather than just the A 280, you can also see if there are any soluble aggregates in your sample by looking for absorbance at 230 nm. This is a quick method and doesn’t require any special reagents, except for the guanidinium, which you may have on hand anyway. Advantages of Measuring Protein Concentration using Absorbance at 280 nm Once you know the absorbance of your protein at 280 nm (A 280), as well as its extinction coefficient, you can use the Beer–Lambert law to calculate protein concentration:Ĭ = molar concentration of protein. Simple but often unreliable, this protein quantification method estimates the amount of protein by measuring the characteristic absorption of the aromatic residues, tyrosine, and tryptophan, at 280 nm on a UV-Vis spectrometer. It can be difficult to decide on the best method for your protein, especially considering that even the most humble protein quantification assay uses some pretty sophisticated chemistry that can trip you up (particularly if you’re working with detergents!). There are several ways to measure protein concentration, and each of them has its own advantages and disadvantages.

Even if you’re doing something more qualitative, having a good idea of how much protein you have will enable you to compare results from one experiment to the next and from one protein to the next. Why is Accurate Protein Quantification Important?Īccurate protein quantification is critical if, for example, you’re trying to determine a binding constant, measure enzyme kinetics, or if you’re preparing samples for a western blot. In this article, we’re going to discuss five major protein quantification techniques-how they work, when they work, and when they don’twork. But there are so many methods out there, how do you know which one is right for you? Accurate protein quantification is key when working with proteins.