You can download ImageJ software (https://imagej.nih.gov/ij/).  Using this software, you can select individual lanes on your gel and then get a color intensity profile across the lane (you'll want to convert your image to black and white probably first).  You can then integrate your protein peak area and divide by the sums of the peak areas for all of the bands in the lane.  Here's a Youtube video that details how to select lanes (they only select a single peak, but the principle is the same) and integrate peak areas:  https://www.youtube.com/watch?v=JlR5v-DsTds

You can download ImageJ software (https://imagej.nih.gov/ij/).  Using this software, you can select individual lanes on your gel and then get a color intensity profile across the lane (you'll want to convert your image to black and white probably first).  You can then integrate your protein peak area and divide by the sums of the peak areas for all of the bands in the lane.  Here's a Youtube video that details how to select lanes (they only select a single peak, but the principle is the same) and integrate peak areas:  https://www.youtube.com/watch?v=JlR5v-DsTds

To calculate an absolute protein amount you have to use an internal sample standard with a known concentration. The Smart Protein Layers technology for 1D gels could be helpful there as there is a specific standard in the sample buffer. With this you can calculate the absolute protein amounts of your protein bands using ImageJ or LabImage. This way you will get absolute amounts not only relative ones.

Hi,

to calculate an absolute protein amount you have to use an internal sample standard with a known concentration. The Smart Protein Layers technology for 1D gels could be helpful there as there is a specific standard in the sample buffer. With this you can calculate the absolute protein amounts of your protein bands using ImageJ or LabImage. This way you will get absolute amounts not only relative ones.

This technology is fluorescence based as fluorescence has a much higher dynamic range. with Coomassie or silver quantification is not possible as their linear range is very low. You come into saturation very fast (not seeing it really) so you can not determine the protein amount by band intensity values anymore!

Thank you  very much dear James Nicholas Vranish and dear Kristin Schulz.

Amended on 20 July 2017

I would like to express my opinion.   PAGE (poly-acrylamide gel electrophoresis) with SDS (sodium dodecyl sulphate or sodium lauryl sulphate) can not become a quantitative method at all.   Hydrophobic proteins usually attach to PAG gel (please see file; IEF for hydrophobic protein), and detection of protein by dye or even by Ag+ is not quantitative (our unpublished observation).   Therefore, we previously used an radioisotope-fluorogaphy method, which is surely dangerous to human health (please see file; Tremella 2D-EP).

Then, HPLC-Surf-SEC protein assay would be recommended to quantitative analysis of the total protein concentration in your purified sample (please see file; HPLC-Surf-SEC protein determination method).

Identification and quantitative analysis of purified protein and contaminated proteins should be utilized with the newly developed proteomics of the PDMD (protein-direct-microsequencing-deciphering) method (please see file; HepG2 Fucoidan).

This PDMD method has recently been successfully applied onto the estimation of contaminated proteins in a commercial lactoferrin (please see file; LF Dr. Kawakami (in Japanese)).   Further, bovine milk also has many proteins from invaded microbes of bacteria, fungi, yeast, avian virus, bacteriophage, and plant at c.a. 17% of bovine milk proteins (our unpublished observation).    Furthermore, human breast milk has bacteria, yeast, and primate's virus, human virus at c.a. 16% of human breast milk (our unpublished observation).   These milks are healthy female origin, and I have previously declared that human healthy organs usually have c.a. 17% proteins from invaded and/or symbiotic microbes (please see also file; HepG2 fucoidan).

Further, I have recently found that human diseased livers have been invaded by microbes (virus and bacteria) at 18.7% in liver of pseudo-cancer (survived), 15.6% in LC tissue (with leprosy; deceased), 25.2 % in HCC tissue (with PBC; deceased), 24.5% in LC tissue (designated as No.6; survived), and 17.9% in HCC tissue (designated as No.6; survived), respectively (median = 21.6%, range = 15.6 - 25.2%: please see file "HepG2 fucoidan").

Although I have not analyzed normal-healthy human livers yet, it seems that levels of invaded microbes seem to be a little higher in diseased livers.

By the way, genetically-engineered crop and foodstuff may additively contain the elevated levels of virus (invaded microbes), and I like the foodstuffs derived from non transgenic crops, fishes, and animals.   Then, I like "natural foods" very much.

If the protein of interest has a heme group, ie it is a hemeprotein, you could check the purity ratio through an electronic spectrum in the ultraviolet and Visible region, making a ratio between the absorbances at 280 nm and the soret band.

I agree with Kou, many papers simply put "processed with Ni-NTA to 95% purity" without further ado, sounds like a number out of a magician' hat.