Tube BSA stock Solution
Tube
1
2
3
4
5
6
7
Pili
Mungbean
BSA stock
Solution
0
5
10
15
20
25
30
Distilled
Water
900
895
890
885
880
875
870
Bradford
Reagent
100
100
100
100
100
100
100
595nm
absorbance
0
0.0965
0.1035
0.1425
0.143
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.
2. Dispense the following into microcentrifuge tubes (three trials
each).
Tube
1
2
3
4
5
6
7
Pili
Mungbe
an
3.
4.
5.
6.
7.
BSA Stock Solution
(µL)
0
5
10
15
20
25
30
2
2
Distilled Water (µL)
900
895
890
885
880
875
870
898
898
Bradford Reagent
(µL)
100
100
100
100
100
100
100
100
100
Vortex briefly to mix contents.
Stand for 5 minutes at room temperature
Read absorbance at 595 nm.
Recoed Abs595
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.
SDS-PAGE
1
2
3
4
5
6
7
Pili
Mungbean
BSA stock
Solution
0
5
10
15
20
25
30
Distilled
Water
900
895
890
885
880
875
870
Bradford
Reagent
100
100
100
100
100
100
100
595nm
absorbance
0
0.0965
0.1035
0.1425
0.143
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.
2. Dispense the following into microcentrifuge tubes (three trials
each).
Tube
1
2
3
4
5
6
7
Pili
Mungbe
an
3.
4.
5.
6.
7.
BSA Stock Solution
(µL)
0
5
10
15
20
25
30
2
2
Distilled Water (µL)
900
895
890
885
880
875
870
898
898
Bradford Reagent
(µL)
100
100
100
100
100
100
100
100
100
Vortex briefly to mix contents.
Stand for 5 minutes at room temperature
Read absorbance at 595 nm.
Recoed Abs595
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.
SDS-PAGE