How do you estimate protein content in osyter mushroom?

Answer 1

ISOLATION OF MUSHROOM PROTEINS

AND

ESTIMATION OF TOTAL PROTEIN BY LOWRY'S METHOD

.

Reagents Required

1. BSA stock solution (1mg/ml),

2. Analytical reagents:

a)50 ml of 2% sodium carbonate (2g in 100 ml of d

)10 ml of 1.56% copper sulphate solution mixed with 10 ml of 2.37% sodium potassium tartarate solution.

Prepare analytical reagents by mixing 2 ml of (b) with 100 ml of (a)

3. Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N) with an equal volume of water on the day of use (2 ml of commercial reagent + 2 ml distilled water)

Procedure

A. Isolation of proteins from Mushrooms

2 grams of mushroom were weighed properly and minced by using 10 ml phosphate buffer supplemented with 2 % SDS (sodium dodecyl sulfate) by using pestle and mortar.The homogenate was filtered through double layered cheese cloth.The filtrate was subjected to ammonium sulphate saltprecipitation method at 65% saturation.The proteins were pelleted by centrifugation and then dried.

C. Estimation of total proteins by Lowry method:

1. Different dilutions of BSA solutions are prepared by mixing stock BSA solution (1 mg/ ml) and water in the test tube as given in the table. The final volume in each of the test tubes is 2 ml.

2. For these protein solutions add 2 ml of alkaline copper sulphate reagent (analytical reagent). Mix the solutions well.

3. This solution is incubated at room temperature for 10 mins.

4. Then add 0.2 ml of reagent Folin Ciocalteau (FC) solution (reagent solutions) to each tube and incubate for30 min at room temperature.

5. By using blank, Zero the colorimeter and take the optical density (measure the absorbance) at 660nm.

5. Plot the absorbance against protein concentration to get a standard calibration curve.

6. Check the absorbance of unknown samples and determine the concentration of the unknown sample using the standard curve plotted above.

Calculation:

Standard calculation:

From standard solution 1mg is 1ml, it indicates 1000 µg in 1 ml of sample, hence 0.2ml contains 200 µg similarly 0.4 ml contains 400 µg of proteins and so on.

Determination of unknown concentration:

From standard graph 0.4 corresponds to X µg of proteins

That indicates 0.5 ml of sample contains X µg of proteins

Hence 100ml of unknown sample contains (X x 100) / 0.5 =----- µg of protein

RESULT:The 100ml of present in mushroom sample isolated contains----- µg or------ mg of protein