Hi.

The answer is yes. It will depend a little bit of how much accuracy you want. If you need only an estimation, you can work by comparison and densitometry to give you a reasonable number. You can use ImageJ software for free to evaluate it.

You will run a new gel including a mass control of the same or similar protein molecular size but with a known amount (mg/mL) and then compare them by densitometry.

You can find some info support here:

http://www.bio-rad.com/en-us/applications-technologies/sds-page-analysis?ID=LW7FGX4VY

Your question has already been addressed in other posts here.

https://www.researchgate.net/post/How_do_you_measure_the_amount_of_protein_in_the_band_after_running_SDS-PAGE

https://www.researchgate.net/post/How_to_properly_quantify_bands_from_SDS-PAGE_by_densitometry

Hope it helps you.

No, you can`t.

Western blotting is/was ever a semi-quantitative method.

Thus, you can say yes or no, regarding the presence of the protein. Maybe, you can say there is more or a lower amount of protein "xy" in sample a vs sample b. Maybe you can estimate there in "much" more or "somewhat" lower protein in sample a vs b. But you can`t say there are 10 µg protein "xy" in sample a or there are 11 µg in sample b. Esimating via band size (visually) is ever a semiquantitative makeshift tool, but do not lead to a reliable quantification (only a qualitative estimation is possible)!

For this purpose you should use quantitative methods, such as ELISA.

Al-Alo K. Z. K. Adriano Costa de Alcantara It is possible on paper, however it will not be a reliable method to determine protein concentration. You run a gel with a protein of known concentration and analyze the intensity of the band densitometrically. Then analyze the intensity of the desired band and calculate the concentration of the protein using the intensity of the protein band for which the concentration is known. One of the problem can be although you are thinking that you are loading the same amount of sample in the wells, but technically it is very difficult to load exactly the same volume in all the wells.

Why are you not measuring the protein concentration spectrophotometrically using Lowry or Bradford method?

Dear Al-Alo K. Z. K.

As you did not tell us everything about your experiment, I can't know how much accuracy you will need. So ....

No protein method is absolute. But take a look at this paper and decide whats is best for you and reasonable for the step you're working on. There are a huge number of papers published using densitometry on gels ... As you may read here, it is an estimate but usually, in my humble opinion and lots of gels performed and evaluated, it goes quite close to the results of other techniques (such as lowry and bradford, fluorescamin, and so on). The use of fluorescent reagents and fluorescent equipments for its detection give even better results as well as with Western Blotting and fluorescent antibodies.

This paper gives a good review of different techniques, read it.

http://chem.winthrop.edu/faculty/grossoehme/link_to_webpages/courses/chem525/resources/protein_quantitation.pdf

As well as these other links below.

https://www.gelifesciences.com/en/us/solutions/protein-research/knowledge-center/western-blotting/quantitation-of-proteins-part-1

https://www.gelifesciences.com/en/us/solutions/protein-research/knowledge-center/western-blotting/quantitation-of-proteins-part-2

https://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/

https://openwetware.org/wiki/Protein_Quantification_Using_ImageJ

http://vlab.amrita.edu/?sub=3&brch=237&sim=1249&cnt=1

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/RP0058.pdf

https://sites.google.com/a/umn.edu/starrlab/protocols/quantitation/quantification-of-protein-bands-using-densitometry

https://www.labce.com/spg1122995_densitometry.aspx

https://www.youtube.com/watch?v=JlR5v-DsTds

https://www.youtube.com/watch?v=6toxQOe4SpQ

https://www.youtube.com/watch?v=nZBsIrP8ljo

Hope it helps.

Hi, Nilay Nandi .

I do agree. But these techniques for protein concentration determination has advantages and drawbacks and none is perfect (bradfor, Lowry, BCA, spectrophotometry, and so on). All of them are giving us their best estimate. The different amino acids bind differently to the binding dyes. So ... Best luck to all of us on our concentrations ... lol ...

As Al-Alo K. Z. K. did not say precise/accurate concentration determination, an estimate amount is fine to lots of different procedures.

All the best.

Bradford methods are never useful for determination of protein concentration in solutions containing detergents. You will get numvers for every measurement, but this will never reflecht your correct protein concentration.

Moreover, visula quantification is ever a subjective and relative way to etimale amounts of proteins and will never reflect the correct amount of any protein (band intentiy will depent on too much variables, concentration of detergents, efficacy of AB binding, affinity to the target and to "non-targets", the experimentator, the transfer rate, quality of corresponding control used ... and so on).

Thus, you should use tested/validated ELISA to get quantities instead of qualitative estimations from WB semi-quantification.

Nope, you can't measure the protein concentration directly from the SDS- PAGE band. But If you go for 2D gel, then you could go quantifying the proteins. You can measure the concentration by analyzing the quantity. Here you need to know the difference between the quantification and concentration.

Dear Dr Andreas.

I need to disagree with your point of view. As you will see, there are bradford assays designed to tolerate detergentes, as below and they work fine.

https://www.thermofisher.com/order/catalog/product/23246

http://www.bio-rad.com/en-us/product/dc-protein-assay?ID=22faf97a-6b8d-4763-8b97-3dc530dcab66

If you did not try, check them and you will see.

All the best.

Sure, but they work with precipitation in the first step or are designed with the precaution to tollerate this source of uncertainty, therefore, Bradford is quite unsuited for this work. Lowry method is established and works well!

For example see:

Pierce™ Detergent Compatible Bradford Assay Kit

Moreover, see Anal Biochem. 1989 May 1;178(2):263-8.

Sensitivity and variability of the Bradford protein assay in the presence of detergents.Friedenauer S1, Berlet HH.

A publication dealing with the issue of opimizing bradfors methods with detergents to get better (not optimal) results fpr protein concentration determination!

The same issue is discussed in vaious other more actual publications, e.g. see Anal Biochem. 2016 Feb 1;494:37-9. doi: 10.1016/j.ab.2015.10.013. Epub 2015 Nov 9. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80. Cheng Y, Wei H, Sun R, Tian Z, Zheng X.

Dear Dr Andreas.

As I mentioned above, those protein concentration techniques are very based on estimations and not on protein quantitation as you will be able to see in lots of important manual like Molecular cloning and all current protocols available. Here I copied and let you all see an example from a webpage about lowry and Bradford, as am example (https://www.coursehero.com/file/p3g2m00/Disadvantages-The-assay-is-not-very-sensitive-and-protein-cannot-be-recovered/).

Although in this example above they present a table saying the compatibility with detergent is only about 0.1%, the Bio-Rad DC protein Assay presents reagent compatibility with SDS of about 10% (which is ten times higher than lowry).

Anyway, as I told before, these techniques are only estimations (some quite close and some not).

Lowry (Folin-Ciocalteu) Assay The color production in this assay arises from the reduction of Cu 2+ ions to Cu + by tyrosine and tryptophan residues under alkaline conditions, coupled with reduction of phosphomolybdate-phosphotungstate complexes of the Folin-Ciocalteu reagent (also referred to as “phenol reagent”) by the reduced copper. The comp lex is detected at 750 nm. This method has a sensitivity of 10 to 200 μg per standard assay. Like the UV method, this assay is often used to follow the purification of a protein where it is important to know the changes in relative protein content but not the absolute amount of protein in the sample. Advantages: The assay is very sensitive, so it requires very little of the protein sample. Disadvantages: The tyrosine and tryptophan contents of proteins vary, so the absorbance per mg of the standard may differ to some degree from that of an unknown protein. Takes time.

Bradford Assay This assay is based on a shift in the absorbance maximum of Coomassie Brilliant Blue G-250 dye (see structure below) when it is bound to proteins via hydrophobic or basic (positively charged) side-chains. The wavelength maximum shifts from 465 nm (dye alone) to 595 nm (dye-protein complex). The sensitivity of this assay ranges from 10 to 140 micrograms in large volume test tube assays and 0.5 to 4 μg in the micro-assays that we will be using.

Advantages: The assay is sensitive as well as rapid. Fewer compounds interfere, compared with the Lowry assay ( Table II ). The assay requires addition of only one reagent. Disadvantages: Proteins vary in hydrophobic character. The dye tends to stick to spectrophotometer cells. The standard curve is not very linear over the usable range.

However, you can`t get protein concentrations from a SDS gel

Before running SDS-PAGE, you have to measure the protein concentration prior sample loading.

Thanks a lot to all the colleagues for these good answers.  I have benefited greatly from these comments and answers.

thank you very much

SDS-PAGE does not allow accurate quantitation of proteins, and you can use NanoDrop to quickly determine protein concentrations. Of course, you can also estimate the protein concentration by the size of the band, but only if there is a standard protein control of known concentration (such as BSA with the concentration of 10 mg/ml).

Thank you