Glass plate (10cm×20cm) was cleaned with ethyl acetate then left for 15 min, 3gm of silica gel was taken in a beaker and 15ml water was added. Then slurry was poured over the glass plate and was allowed to dry.When the plate was dried ,heated by hot plate for few minute. Then protein was spotted on a preparative slide.
Preparative TLC for isolation of antioxidant protein
TLC was run in solvent BAW(4:1:1), silica was scrapped from Rf 0.25 to 0.30, Protein was eluted in glycine buffer.
Test for protein estimation
Small amount of plant extract powder mixed with few amount of glycine buffer,Then filtered it to a appendrop tube,Kept it inside refrigerator,Then prepared all the reagents required for protein estimation.
Preparation reaction mixture
a)2%Na2co3(0.1N NaOH)-solution A
b)1%CuSo4(in distilled water)-solutionB1
c)2%Sodium Potassium Tartarate(in distilled water)-solution B2
d)Reagent=A:B(49:1) (B=1mlB1+1mlB20)
4 clean test tubes were taken.1ml of distilled water and 4ml of AB was added to each of the 2 test tubes and it was allowed to incubate at 37ºC for 30 minutes after which 0.5ml of folin reagent was added and incubated for 30 minutes at room temperature. These two test tubes served as blank. Then 20µl sample+980µl DW and 4ml of AB was added in third test tube then it was allowed to incubate at 37ºc for 30 minutes after which 0.5ml of folin reagent was added and incubated for 30 minutes at room temperature. Then 40µlsample+960µldw and 4ml of AB was added in 4th tube then it was allowed to incubate at 37ºc for 30 minutes after which 0.5ml of folin reagent was added and incubated for 30 minutes at room temperature. Then OD was taken at 510nm.
Calculation of % of pure protein
% of pure protein in crude protein =
Mean OD of pure protein sample / Mean OD of crude protein sample × 100
Sanjeet KumarNBPGR, CRRI Campus, Base Centre cuttack.
