There is no standard for this. It will depend on how much His-tagged protein was present, how well it bound the column, and how much was collected in the elution.

It sounds like something in your elution buffer is interfering with your protein assay. You can try a different protein assay, such as Bradford, or TCA-precipitate your samples to remove interfering substances before doing the protein assay.

Run your fractions on SDS-PAGE to select the ones with your protein. Pool the best fractions, dialyze the sample to remove imidazole and interfering substances. Then quantify the protein.

Dear Danielle

Did you mean, if you load 20mg of total proteins?

As Adam suggest in this case There is no standard for this because It depend on how much His-tagged protein was expressed, how it bind to coloumn (not ever the His is well exposed and the protein bind) and how the wash were able to remove impurity and this can depends for different factors:

1) THe amount of the resin that you use. Low amount of resin can result in resin saturation and incomplete recover of your protein but generally it result in higher purity level because your protein will compete with the impurity. High resin amount can increase the His-protein total recovery but also increase the impurity.

2) The volume of the washing that you applied before elution. Generally with 20mM of imidazol you need to do long wash (at least 20CV).

3) Which lisis buffer did you use. some buffer or lisis approach more strong are able to extract more low soluble proteins and the final purity will decrease. 

for quantification you can use Bradford and BCA assay. Generally they work well with elution from imac. 

I do not have experience with lowry but 280nm  after 1 imac purification step can be highly affected form the presence of DNA. Did you used DNAse in the lysis buffer? Which 260/280 ratio di you obtain in your quantification? 

Ciao

Manuele

"Blank using elution buffer gave higher values than samples" is simply not possible if you are following absorbance at 280 nm.

TCA preciptation is a good idea, thank you. I'll try bradford too. I ran a SDS-page with BSA standart and now a have a idea of how much protein I have.

Manuele I whased with 25CV. If a use a higher imidazole concentration, can I use less CV to whash?

I didn't use DNAse.

Do you have a protocol of 280nm assay? This so many different ones in internet that I don't which to use. It's my firsr experience with protein.

Omar, Absorbance does not lie.

Dear Amaral

regarding the washing buffer. You can try to increase imidazole concentration ar 40 or 60mM but in some case it can also detach your protein, therefore i suggest to you to maintain the 20mM step and if you want try to increase the final purify try to add an additional washing step using 40mM and store all the fractions at 4°C until you run the SDS-page of all. Therefore if at the end in the SDS page you will found your protein also in the 40mM wash, you can dedice to pull it with the elute. 

Regarding the 280nm protocol

Sone more question :

Which kind of instrument are you using to measure it? I need to know it for suggest to you a protocoll.

A standard Uv/vis spetrophotometer with cuvette or a microvolume spettophotometer as nanodrop?

In the first case are you using quartz cuvette or plastic cuvette suitable for reading in the Uv (eg BRAND UV cuvettes - Z637939 Sigma aldrich) or standard plastic cuvette suitable for OD reading??

Because standard plastic cuvette are not transparent at 280nm and using it also water can give you very high adsorbance signal and you will not be to do an accurate blank with any buffer. And as Omar told, the fact that the blank supply higher signal that the blank is very strage

. However if you are not using the correct cuvette and you are in satuation (Absorbance higher than 1) is it possible that  the addition of the sample do not incrrease the value because the instrumenti is saturated and give you causal response.

If you are using the correct cuvette.

Is important that you are not just reading the value at 280nm but you acquire the scan and you observe the curve shape.

The sample subctracted for the blank if contain a purified protein need to show you a peak with a maximin at 280nm. If you observe a peak but with maximun at 260nm, it is contaminated by DNa and also if you are able to observe protein in SDS-page you cannot use the value at 280nm to quantify it because it is derived from the sum of value of DNA and value of protein.

In this case you can try to prevent it by adding dnase in you lysis buffer or to remove if after elution but before quantification by performing some cicle of  concentration/dilution by ultrafiltration and hope that DNA fragment pass trhough the membrane. 

However remind that in general you need to dilute your sample to obtain a final 280nm signal lower than 1 and that blank with signla higher that 1 are strange and indicate that there is something wrong in your procedure.

ciao

Manuele

Manuele, thank you so much! You're helping me a lot! I use varioskan flash (thermo), but I read the samples in a plastic 96 well plate. But I was dumb becaude I know that in a spectrophotometer I must use quartz cuvette. Thank you! I run a SDS-page with 2, 10 and 20 micrograms of BSA (last tree lanes).

i can use a nanodrop (thermo). In this case should I dilute the sample too? How much! 1:1000?

It was in lowry assay that my blank with 500mM of imidazole gave a higher absorbance than samples. I know it's very strange but is not impossible, since I saw It. Maybe I did something wrong.

I think that in my eluted fraction I do not have same imidazole concentration that in elution buffer,, since it get attached to the column. Maybe thats why Blank had a higher absorbance than samples. Am I wrong?

thank you!

So probably you need to use a UV traspatent plate, that generally are used for quantification of DNA samples.

GReiner UV star (SIGMA cod. M3812) can be ifne. 

Remind that in the plate the optical lenght is not  fixed =1cm  as in the cuvette but it depends from the sample volume, therefore once you want extrapolate the sample concentrarion from the lambert-beer law you need to know not only the protein estinction coefficient that could be calculated with programs as prot param tool, but also measure the sample thickness that depends from the volume that you insert in the well. 

good luck

Manuele