protein lab
DATE: 11/10/12
COURSE CODE: BIOL 2365 Comparative Biochemistry
TITLE: Proteins and Amino Acids
RESULTS:
Table 1: The results of experiment 1; the Lowry Test
Volume of Standard Protein/ Unknown (mL)
Absorbance at 750 nm
0
0.000
0.1
0.017
0.3
0.135
0.3
0.155
0.5
0.230
0.7
0.323
0.7
0.310
1.0
0.457
1.0 Unknown 1a
0.463
1.0 Unknown 1b
0.433
1.0 Unknown 2a
0.237
1.0 Unknown 2b
0.159
Table 2: The results of Experiment 2; Ninhydrin Test
Amino acid
Color
X
Bright yellow
Y
Dark yellow
Z
Light purple
Table 3: The results of Experiment 3; Protein Precipitation
Precipitant
Observation
TCA
White fluffy precipitate in large mass, the fluffy mass goes from the bottom to the top of the tube. The solution was thin and loose.
Ammonium sulphate
White gel-like precipitate, the solution was thick.
HCl
White precipitate accumulated at the top and also the bottom of the solution. The solution was thin.
NaOH
No precipitate seen, the solution was cream in color.
Copper sulphate
Light blue coagulated precipitate seen at the top of the solution.
Lead Acetate
Cloudy solution with small specks of white precipitate.
Ice cold ethanol
Cloudy at the bottom and about 0.5 cm of the top of the solution was clear.
Table 4: The results of Gel Filtration Chromatography
Eluate
Component
Volume collected in mL
First
Blue dextrin
3.00
Second
Brown cytochrome
7.00
Third
Clear phosphate buffer
5.00
Table 5: The result of Electrophoresis of Amino Acids
Distance moved (+/-) cm
Amino acid
10% Acetic acid
Phosphate
NH4OH
Glycine
-6.0
-3.2/+3.0
+9.0
Lysine
-7.0
-2.0/0
+3.7
Glutamic acid
-4.5
-3.2/+3.0
+5.0
SAMPLE CALCULATIONS:
(1) Protein concentration of Standard Protein Solution:
1mL = 200µg protein
Therefore 0.1mL= 0.1/1× 200 = 20µg
(2) Protein concentration of the two unknowns:
Gradient of the line = Y2-Y1/ X2-X1
0.135-0.017/
COURSE CODE: BIOL 2365 Comparative Biochemistry
TITLE: Proteins and Amino Acids
RESULTS:
Table 1: The results of experiment 1; the Lowry Test
Volume of Standard Protein/ Unknown (mL)
Absorbance at 750 nm
0
0.000
0.1
0.017
0.3
0.135
0.3
0.155
0.5
0.230
0.7
0.323
0.7
0.310
1.0
0.457
1.0 Unknown 1a
0.463
1.0 Unknown 1b
0.433
1.0 Unknown 2a
0.237
1.0 Unknown 2b
0.159
Table 2: The results of Experiment 2; Ninhydrin Test
Amino acid
Color
X
Bright yellow
Y
Dark yellow
Z
Light purple
Table 3: The results of Experiment 3; Protein Precipitation
Precipitant
Observation
TCA
White fluffy precipitate in large mass, the fluffy mass goes from the bottom to the top of the tube. The solution was thin and loose.
Ammonium sulphate
White gel-like precipitate, the solution was thick.
HCl
White precipitate accumulated at the top and also the bottom of the solution. The solution was thin.
NaOH
No precipitate seen, the solution was cream in color.
Copper sulphate
Light blue coagulated precipitate seen at the top of the solution.
Lead Acetate
Cloudy solution with small specks of white precipitate.
Ice cold ethanol
Cloudy at the bottom and about 0.5 cm of the top of the solution was clear.
Table 4: The results of Gel Filtration Chromatography
Eluate
Component
Volume collected in mL
First
Blue dextrin
3.00
Second
Brown cytochrome
7.00
Third
Clear phosphate buffer
5.00
Table 5: The result of Electrophoresis of Amino Acids
Distance moved (+/-) cm
Amino acid
10% Acetic acid
Phosphate
NH4OH
Glycine
-6.0
-3.2/+3.0
+9.0
Lysine
-7.0
-2.0/0
+3.7
Glutamic acid
-4.5
-3.2/+3.0
+5.0
SAMPLE CALCULATIONS:
(1) Protein concentration of Standard Protein Solution:
1mL = 200µg protein
Therefore 0.1mL= 0.1/1× 200 = 20µg
(2) Protein concentration of the two unknowns:
Gradient of the line = Y2-Y1/ X2-X1
0.135-0.017/
References: Amrita University. Qualitative Analysis of Amino Acids.2012. http://amrita.vlab.co.in/?sub=3&brch=63&sim=1094&cnt=1 (4 October 2012) Boyer, Rodney. 2012. Biochemistry Laboratory. Pentice Hall Publishers, New Jersey. Nelson, Cox. 2008. Leningher Principles of Biochemistry. Fifth edition. W. H Freeman and Company, New York. Nigam Aarti, Ayyahguri.2012. Archana Lab Manual in Biochemistry, Immunology and Biotechnology. Tata Mc Graw Hill Publishing Company. pHscale. Net, pKa and Ka, 2012. http://www.phscale.net/pka-ka.htm (13 October 2012). University of California, Department of Chemistry. 2011. Chemistry 2B Laboratory Manual. University of California. Davis, CA.