Tube BSA stock
Solution DistilledWater BradfordReagent absorbance595nm
1 0 900 100 0
2 5 895 100 0.0965
3 10 890 100 0.1035
4 15 885 100 0.1425
5 20 880 100 0.143
6 25 875 100
7 30 870 100
Materials and Methods 1. Protein extraction
-deflatted legume seed meal of of munfbean and pili -extraction buffer which consisits of
0.40 M NaCl in 35 mM potassium phosphate buffer 10mM of β-mercaptoethanol
Sodium azide
pH 7.6 with 0.10 mMPMSF (Phenylmethylsulfonyl fluoride) 2. Protein Content determination:Bradford method
-seed crude protein solutions -biorad protein dye
-protein molecular weight markers 3. SDS-PAGE
a. Extracted protein solutions
b. Protein molecular weight marker c. Acrylamide solution
d. Separation gel buffer e. Stacking gel buffer f. 10% SDS
g. Ammonium persulfate(APS)
h. TEMED (Tetramethylethylenediamine) i. Tank buffer
4. Gel Filtration PROTEIN EXTRACTION
1. Weigh 50 mg seed meal samples and place in a 1.50 mL microcentrifuge tubes.
2. Add 1.0 mL extraction buffer (SDS-PAGE separation buffer). 3. Vorter for 2 minutes.
4. Centrifuge filtrae at 10,000 rpm for 10 minutes at 4 degrees
5. Collect supernatant into fresh tube. Keep in ice bath or refrigerate until use.
PROTEIN CONTENT DETERMINATION Preparation of Standard Curve
1. Prepare 5.0 ML stock solution of BSA (Bovine Serum Albumin) at 1mg/mL distilled water.
Tube BSA Stock Solution
(µL) Distilled Water (µL) Bradford Reagent(µL)
1 0 900 100
2 5 895 100
3 10 890 100
4 15 885 100
5 20 880 100
6 25 875 100
7 30 870 100
Pili 2 898 100
Mungbe
an 2 898 100
3. Vortex briefly to mix contents.
4. Stand for 5 minutes at room temperature 5. Read absorbance at 595 nm.
6. Recoed Abs595
7. In graphing paper, plot the absorbance (y-axis) versus
concentration of the standard solution (x-axis). Label the axs properly stating the units of measurements used.
8. Determine the ‘best fit’ straight line among the set of x, y data pairs.
9. Perform linear regression analysis. Ther acceptable r value ranges from 0.9-10.