I want to subject a seed to different processing methods to reduce anti-nutrional factors present. Can I use SDS-PAGE Electrophoresis to characterise how the processing methods (Cooking, roasting, sprouting and soaking) influence the protein content. Please, I need references. Thank you.
SDS-PAGE, or even better 2D-electrophoresis, is a simple way to look at the overall composition of proteins in a sample. The individual spots can be identified by mass spec proteomics techniques. In seeds, I would guess some sample pre-processing would be needed to reduce the starch and fat content. Also, if the seeds contain one predominant storage protein, it may be difficult to see anything else on gels but that one protein.
If you are interested in knowing whether the processing methods denature proteins, SDS-PAGE alone won't tell you because you have to denature the proteins anyway (with SDS) to run the samples on the gels.
Thank you for your response Adam B Shapiro. How best can I then establish the quality and quantity of protein in the processed seed - in addition to using amino acid scoring and proximate analysis?
And, I was thinking, please correct me if I am wrong, won't I notice variation in expression of protein on SDS-PAGE - ladder - with different processing methods imposed on the seed?
Thank you.
If you want to use SDS-PAGE for your protein, the before loading your protein samples, you need to denature the protein by adding sds sample loading buffer and heating it at 95-100Deg. Sample loading buffer contains DTT or other protein denaturating agents. SDS-PAGE is not good technique to check protein quantity and quality. You may use LC-MS/MS for protein identification and quntitation.
SDS-PAGE, or even better 2D-electrophoresis, is a simple way to look at the overall composition of proteins in a sample. The individual spots can be identified by mass spec proteomics techniques. In seeds, I would guess some sample pre-processing would be needed to reduce the starch and fat content. Also, if the seeds contain one predominant storage protein, it may be difficult to see anything else on gels but that one protein.
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