I am looking for simple protocols for total protein extraction or phaseolin extraction from bean seeds so that expensive materials like DTT or PMSF does not need to be used. Any suggestions?
A cheap solution and usually a good proxy for total protein concentration could be the Bradford assay.
thanks a lot for answer to my question but I am looking for a simple method to protein extraction. Bradford assay is a method for determination of total extracted protein concentration.
Follow the procedure of using saline phosphate buffer (PBS) for protein extraction.
Dear Soufian
Extraction of proteins from plant material requires the existence of protease inhibitor, at least one. PMSF inhibits serine proteases which are the main class of proteases in most plant tissues. But it is expensive and also requires great caution because it is highly toxic to human. If you do the extraction without a protease inhibitor you will lose some of your proteins and in case you ran them on a gel, you will get a streak rather than protein bands.
I would suggest that extract your proteins with buffered SDS. SDS will stop all enzymes by denaturing their proteins. You can use this extract for SDS-PAGE but you cannot use it for protein assay by Bradford method because SDS forms a blue colour with Bradford reagent. If you want to assay your proteins in this extract, you will need to use Lowery method which does not cost too much.
Good luck
A simple method for protein extraction from plant material is based on solubility. Water will extract the water soluble proteins; 5% NaCl will extract globulins; 70% ethanol will extract prolamins while 1% NaOH will extract glutelins. This means that it depends on the type of protein you are looking for. Phaseolin is a globulin protein and most of it will be extracted using dilute saline (5% NaCl)
Dear Philippa Ojimelukwe
Your answer is interesting, do you have an article in which these procedures are detailed. This will help me.
The standard seed storage protein extraction is to extract first with water to remove the albumins, then a salt solution to remove the globulins (phaseolin), then alcohol to remove alcohol soluble proteins and finally NaOH to remove the "glutelins". Are you using immature or mature seeds? There are lots of references on this from the 1970s. Dr. Niels Nielson was one the experts in this field as was Robtert Goldberg.
Which solution is used to extract protein?
Can be used Lowry method for protein extraction from seeds?
No, the Lowry cannot be used to extract the protein from the seed. You need to do the sequential extractions that I described above. Then an aliquot of your extract is used for protein determination with Lowry. You will probably have to dilute your sample to get an accurate reading with the Lowry's. For the Lowry assay you will need to make a standard curve of know protein concentrations. Usually bovine serum albumin (BSA) is used for this.
I shall check and get back to you. Be kind enough to remind me if i don't in the next one month. I am a little very busy now.
Best regards Philippa
I used the method of sequential extraction as stated by Philippa but my pellet obtained is very less and its protein content is too low in ug/ml; can you suggest what to do increase the yield?
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