I have realised that a) my calibration was BGG not BSA and b) that all I need if I understand correctly is to appy the BL equation to my absorbance at 280 nm value and as the extinction coefficient is published at 0.1% this will already provide a mg/ml concentration for my protein. Please feel free to add any thoughts as my next thought is.....if i used the Bradford reagent which absorbs at 595nm will my protein in isolation be concentrated enough to view at 280 nm?

Dear Marie,

usually the sensitivity of UV is comparable with that of a Bradford assay. But be aware that every method has its inherent compatibility problems. So for example, if you have some impurities that are not from our protein but absorb at 280nm, they will distort your measured concentration.

But if UV works for you, it has the advantage that you will usually not lose protein for quantification.

In our lab and with our collaboration partners we usually stick to one method for reproducibility reasons. Therefore I suggest to find out whicht method is most convenient for you and stick to it.

I hope I could help you a little bit.

Best,

Lukas

PS: You can find a nice overview here: http://www.labome.com/method/Protein-Quantitation.html

Thank you for your very helpful answers Lukas and Sven. As you say Lukas it is best for me to apply a consistent approach for reproduciblity so I am going to try a comparison using my own protein @280 and then with Bradford reagent and then make an informed decision from there. I had a look at the link you posted too thank you, and good to realise that each system has its limitations. I had not considered forward application of my protein as i have previously only used Bradford reagent and so that is an added perspective, thank you.

Thank you Charlotte. As a starting point I wanted to look at how I could use Extinction coefficent at 280 compared with results of Bradford Assay. So I did so yesterday with commercial standards of BGG and obtained a perfect linear curve with Bradford but a totally anomolous result at 280 nm using the ECpercent (1.4). Do you have any thoughts as to why that may have happened?

Thanks for the link Charlotte, unfortunately I dont have access to the full text, cheers anyway!

I was using BGG to establish the protocol and decide on which method to use with known purity and concentrations, before going on to use my own protein. The results of A@280 were inconsistent with differing concentrations showing similar absorbance values. A colleague has mentioned today that the problem may have arisen from the cuvettes I used, and has advised I use only quartz ones at 280 nm. All of the values were also below 0.1. I am going to try again with the quartz cuvettes. I will be carrying out an extraction with my own protein in the next few days, so am glad to be overcoming these initial problems before I start with my precious samples! At this stage with the BGG I calibrated with dH20 and used this as a blank for A@280, and for the bradford assay i used the dye plus dH20 as a blank and subtracted the absorbance from the rest of the assay values. The reason I used dH20 is that my protein is eluted from affinity column with this.

cheers, Marie

the elution process, is off an affinity column,crude plant extract is applied, then the first wash is PBS followed by a second of NaCl til absorbance at 260 mn falls below 0.001 at which point target protein is eluted with dH20, does that sound more reasonable? I was working from an early protocol in another paper. Thanks for your input! I will also look out for the disposable UV cuvettes as you say they will be easier to work with.

thanks again.

Here is Charlotte's suggested paper in pdf !!

Opps!!! Here it really is:

Some points:

BGG= bovine gamma globulin ????

A. Why don't you measure the A280 of a 1mg/ml BGG itself. Don't dilute it or anything. You should get a high value. From this see if the E (epsilon value) is in the expected value. This is the best test to run first. 1mg/ml of BGG should be 1.3 to 1.4 A280.

B. Don't dilute your BGG too much. The A280nm is not very sensitive for dilute proteins.

C. What is BGG dissolved in? If it is from Pierce it is in 0.9% NaCl. Many buffers will also absorb at 280 nm. You need to blank (zero instrument) against same buffer as BGG is in.

D. If the protein is dilute than at any concentration the absorbance of buffer will be highest. The phenomenon of a 0.1 reading no matter what suggest that. If BGG is in some buffer than diluting it into water will also give you buffer absorbance. The constant 0.1 value means BGG is too dilute. Go to step A.

1. Absorbance at 280 does not require a standard curve - with the E value known. It is a direct application of Beer Lamberts Law.(E=epsilon).

2. What is the E value you have? The actual number. Low E values mean small number of Trp,Tyr etc residues.

3. Stick with quartz cuvettes until you have everything the way you want.

4. The Bradford method is sensitive to protein amino acid composition. It is not too accurate, and best used for crudes. A better assay is the Pierce BCA assay, where protein-to-protein variation is much lower.

Good luck !

Thank you Prem, the paper is very useful! so, today I have obtained quartz cuvettes and taken up as many of all your suggestions as possible. Just concentrating on the bovine gamma globulin standard for now so that I know that I am not introducing systematic errors into my work. I prepared a 0.9% NaCl blank as you pointed out Prem, and calibrated against that. I used undiluted BGG at 1mg/ml concentration. with and E value of 1.4, which gave A@280 of 0.103 resulting in a value of when I applied Beer Lambert equation (if I am doing so correctly!) of 0.7 which falls far short of the actual concentration of 1mg/ml. My own protein has a previously published EC of 2.08. Your help with this everyone has been so valuable!

Charlotte, I will ultimately be doing structuralwork with the protein but the priority for the next few weeks is to gain a thorough understanding of the magnitude of yeild from the natural product and to test the extraction process robustly. Can i ask what your thoughts about the dH20 as an elutant are? I am at the very beginning of my research degree so excuse my lack of understanding!

You have a problem with the spectrophotometer. A 1mg/ml solution can never give an absorbance of just 0.1. Everything you run on the machine is 0.1 !!

1. Is the spectrophotometer set on UV LAMP?

2. Make sure you are not using the VISIBLE lamp ...that is ordinary lamp light - used for wavelengths in the visible range

3. Does your spectrophotometer have the option to choose visible and UV?

4. Finally, is this really a UV spectrophotometer, and not just on for use at visible wavelengths. Many specs are just plain visible range because UV is more expensive, and visible ones are used for monitoring routine bacterial growth.

This is a VERY common error.

Also, here is very useful tech sheet from Pierce.

Thank you Prem, I have the EC pdf most useful! I am afraid to say I assumed the spec and will definitely need to check tomorrow. As an undergraduate I used it for the Bradford Assay but as that is at 595nm that is why I obtained the perfect linear curve!! Feeling rather silly but extremely grateful!! Thank you! I have checked the manuals available online and it would suggest that the visible range only machine cannot be set below 320 which my machine can do - it automatically measures at 260 & 280 so will check wether it has lamp settings which can be switched on or off as soon as I can. Thanks for your guidance!

What model is it?

Hi Prem, it is a jenway Genova MK3, and your help has enabled me to locate a problem with the unit itself. I obtained the manual which has identified a lamp issue, so I am starting fresh with a new machine. full of optimisim :)

Hi Charlotte, thank you for your advice. I have previously extracted this protein in water, having followed a previously published protocol, and then conducted bioassays with a free living nematode to exam the toxicity of the protein. It appears to retain function throughout a number of biological tests. I conducted that research as an undergrad and am now doing my PhD on it so wish to understand the protein in a much more indepth way. The reason i wished to move to measuring at 280 nm was to avoid the use of the Bradford reagent/BGG standard curve in order to increase speed and accuracy of concentration determination using the extinction coefficent i now have access to which i did not have as an undergrad. Thanks for your help.