I want to extract proteins and carbohydrates from microlgae for quantitative estimation. For proteins I am using a Bradford Assay while for carbohydrates I am using an Anthrone assay.
Do you want to have an approximative or exact estimation of the proteins? The Bradford is convenient for an approximative determination by using a low amount of proteins, however some natural pigment present in microalgae could interfere. In order to have the best estimation of the total amount of protein is the use of the old methods (biuret, Nitrogen etc...) which need a lot of proteins being previously separated from other cell components. I don't know if the method for determination of carbohydrate is also working with the polysaccharides.
The sonication method to break microalgae cells should be likely working. The use of French press wass the best way to obtain an excellent enzymes activities in cell extract. The use of small sized glass beads could also be useful
Good luck
@Robert: Actually the amount of proteins is very low due to very less biomass of different algal species. And yes Bradford is convenient that is why am thinking to use it. For sonication lysis buffer contains:10mM Tris HCl + 5mM EDTA + 1% Triton X 100 + 1mM PMSF + 2mM DTT.
I haven't had much success using sonication. I know that many algae have strong cell walls and I assumed this was the reason. If you have access to one, a French Press is an excellent way to generate a homogenate. TCA precipitation of the protein prior to measurement will help improve accuracy. Also, unless you have a protein standard that matches your target protein, I wouldn't use the Bradford method. That method is strongly dependent on the composition of the protein.
I am not sure about proteins. Microwave technique has shown better results for microalgal cell disruption for lipid extraction. Protein denaturation could be concern while using microwave.
Hi Uday,
Are you referring to chlorophytes or cyanophytes? We are working on cyanobacteria and we use lysis buffer along with homogenization using glass beads. We have good success with that. For estimation Bradford is very much ok. For carbohydrates we are using HCl hydrolysis method. Estimation by Anthrone.
Gud luck.....
Anthrone assay for total carbohydrates has got several disadvantages so it might be better to use phenol-sulphuric acid method descibed by Dubois et al. (1956) in ''Colorimetric Method for Determination of Sugars and Related Substances''.
@Dinesh Babu Gnanasekaran, Thanks for suggestions. I think HCl hydrolysis method will be good for extraction of carbohydrates.
@ Lukasz Wojciech Wajda
Can you please tell me the disadvantages of Anthrone assay for total carbohydrates.
Dear Mr Singh, I am sorry for my late response. Following Dubois et al. (1956): ''(...), anthrone reagent is expensive and solutions of it in sulfuric acid are not stable. The anthrone method also suffers from the disadvantage that, while it is satisfactory for free sugars and their glycosides, it is of limited use for methylated sugars and the pentoses.''
Lowry's method in combination with Lopez et al., 2010 can also be fruitfull for the extraction of proteins from microalgae.
Total extraction and quantification of carbs is a Mission Impossible. Total carbs are usually estimated by difference once you know the dry wight of your material. It really depends on the goal of your analysis and how much time/effort you can afford. Rupturing the cells will help, just not sure about the error that will be introduced by the interferences present in the whole extract. Phenol-sulfuric test will only give you an estimate. Every sugar has a different extinction coefficient.
Again, if you know the dry weight and have decent values for lipids, proteins plus ash; then the difference accounts for total carbs.
Alone sonication is not best method for extraction of proteins but it is good method for cell lysis and solubilzation of proteins followed by purification steps. As per quantification of purified proteins Bradford and Dry weight can be optimized for more accurate results.
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