I would like to quantify a recombinant protein (with native signal sequence) secreted by Pichia pastoris using the SDS PAGE. Since my standard protein (plant origin) is not pure, I would like to use a known concentration of a protein with a similar molecular weight to quantify the bands accurately. Would this be an ideal way to quantify the protein?
I am not sure SDS-PAGE is the right method of quantification of protein. It could give information about the purity, molecular weight and size of a protein. It could give information about the polypeptide composition of the protein and is currently used in peptide mapping. I am not familiar with its application in protein quantification. For the problem at hand, I suggest other methods like densitometry after SDS-PAGE, or simpler methods like Bradford method or UV absorption.
I wouldn't exactly call it "quantify", but you can get a decent estimate. Pick one or a few standards with similar Mw, and amino acid sequences that aren't too exotic. Might use imageJ to quantify the bands.
Hi there,
If you use Comassie Blue, intensity of the staining will depend on the actual aminoacid content of the protein (mainly basic aa..). So quantification will be approximative just like protein assays like Bradford, because based on staining using a given protein as a standard.
Hi Jewel,
I recommend you to prepare multiple dilutions of a known protein (e.g. BSA) and load these on a gel preferably in increasing order of concentration; and then have your recombinant protein of unknown concentration, loaded right next to these (in triplicates may be). Followed by this, you will do a Coomassie or silver staining after electrophoresis and then quantify bands using Image J software. You will be able to obtain a rough value for your recombinant protein concentration just by eyeballing known and unknown protein bands and from Image J. However, if you wish to increase the accuracy of your results, make a standard curve out of the known protein by having those in triplicates or quadruplicates on a gel followed by band quantification of all. Using this curve you can estimate the concentration of the unknown protein.
I am not sure if a known protein of similar molecular weight is required for the problem you are trying to address here.
All the best!
Anoop
John Schloendorn Dominique Liger and Anoop Arunagiri thank you very much for the detailed answers.
John Schloendorn I am using Image Lab software to quantify the bands. If I can calculate the unknown band using a known band, wouldn't that qualify as a quantification? (image attached)
Dominique Liger Is it possible that I estimate the protein content directly from a stain-free technology i.e., after activating the stain-free gel containing the protein and quantifying the bands using the Image Lab software right after the electrophoresis step?
Anoop Arunagiri Thank you for the detailed explanation. I will try as per your suggestion. Do I need to perform blotting after each electrophoresis to confirm the quantity of the unknown protein?
Hi Jewel Ann Joseph
If you have an antibody that can recognize your protein of interest, then it is good to do a western blotting; however that will not provide additional information on the protein quantity. A Coomassie or silver staining (followed by band quantification) is sufficient to help you get a rough estimate of your protein concentration.
Anoop
I am not sure SDS-PAGE is the right method of quantification of protein. It could give information about the purity, molecular weight and size of a protein. It could give information about the polypeptide composition of the protein and is currently used in peptide mapping. I am not familiar with its application in protein quantification. For the problem at hand, I suggest other methods like densitometry after SDS-PAGE, or simpler methods like Bradford method or UV absorption.
SDS PAGE is one of methods for identify to presentation of protein and peptide, but due mentioned amount of ladder, it were used for approximatly quntification amount of protein, but not suitable methods for your goal.
please more search and try other methods in mentioned other answer such as BSA, BCA kit, bradford assay and etc.
As all suggested, you can do a quantitative estimation via densitometry using standard protein of know concentration as reference. All gel documentation system software have densitometry option. Load your standard protein of know concentration in 3-4 dilution in 20-1000 ng range. select your band of interest and follow software instruction.
If you don't have gel doc than after staining, you need to scan your PAGE and can do pixel quantification of standard and than can calculate yours samples as well.
Few suggestion:
1. Never load high amount of protein, in that case your band will be over saturated and you will end up with wrong estimation.
2. Always load your unknown protein in 3-4 dilution, calculate individually and than do a average to get a estimation closer to correct one.
3. Always ask 2-3 lab members to estimate PAGE individually and calculate a average figure, as just band size selection variation can give miscalculations.
4. Always check, if you have a background reduction option in your software, just apply that for better results.
Best wishes.
Vikas
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