I am having difficulty finding the protein from yeast? Just want to extract protein and detect it by biuret reagent methods. I stored my yeast samples at -20C.
So my question is how can I rupture the yeast cell wall? and It is possible to use only 1N NaOH to rupture the cell wall and use the biuret reagent method to quantify it?
Would really appreciate your help. Thank you
I believe it is common to use a bead beater to break open yeast cells.
Is your protein an unknown protein. Give a name and a possible location. Just a little guide.
Olusegun Oshibanjo In all of these article they use other chemicals?
My question is why do we need to use other chemcial if we can rupture the cell with NaOH and glass beads?
Rupter the cell and find the total protein with buiret reagent method? Sounds logical?
Do you know the location of your protein of interest? Or, you haven't chosen yet what protein to work with? The glass beads method is appropriate, but you need to add a cocktail of protease inhibitors to avoid protein degradation. Keep them cold all the time. As for the protein assay, you can compare different methods here: https://www.thermofisher.com/ec/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-assays.html
Francisco Javier Alvarez : Thank you for your response. At this stage, I just want to find the total protein present in the yeast. What I understand is that I can used glass beads to rupture the cell (while doing it, Cool it down regularly) and use biuret reagent method to measure the total protein content.
I don't need to add any chemicals for cell lysis.
Maurice Ekpenyong : Thank you for your response. I just want to find the total protein in yeast.
Dear Obaidullah Mohiuddin, there are chemicals that help lyse the cells properly. The buffer I used is called TNE and it contains the following: 0.876 g NaCl, 5 ml of 1M Tris-HCl (pH 7.4), 2.5 ml of 0.2 M EDTA and water in a volume of 10 ml. In my research I bashed the cells with glass beads 2 x 45 sec, placing the sample 3 min. on ice in between. The cocktail of protease inhibitors I used contained (100X): 0.0174 g PMSF, 0.0392 g benzamidine, 0.002 g of Pepstatin A and the rest was DMSO for a volume of 1 ml. I share with you my publication in case you find it useful: Article Identification of an N-Acetylglucosamine Transporter That Me...
Obaidullah Mohiuddin In some case, chemicals are better to maintain buffer condition and inhibits protease activity.
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