Alabama A & M University Protein Electrophoresis Biochemistry Lab Report For Lab 3, the videos do not have any data. Below is an SDS-PAGE result that you w

Alabama A & M University Protein Electrophoresis Biochemistry Lab Report For Lab 3, the videos do not have any data. Below is an SDS-PAGE result that you will use for your assignment. For the procedure-see the attached documents. Assume the protein concentrations are 2 mg/mL for the known proteins. For determining the weight of the unknowns, make a calibration curve from the MW ladder (plot log(MW) vs distance migrated for a y=mx+b line of best fit), estimate MW of myo and cyt C, how does it compare to lit values? And what proteins do you think unknowns A and B are based on size. OBJECTIVE:
Separate a series of standard proteins by SDS-PAGE and create a standard
curve (calibration curve). Use the standard curve to determine the molecular
weight of unknown proteins.
MATERIALS:
1X Tris Glycine/SDS Running Buffer (prepared from 5X Running Buffer)
Sample Buffer
62.5 mM Tris-HCl, pH 6.8, 2% SDS
0.1% glycerol, 0.05 % 2-mercaptoethanol, 0.05% bromophenol blue
Pre-cast 12% polyacrylamide gels
Table 4.1:
Protein
Phosphorylase b
BSA
Ovalbumin
Carbonic anhydrase
Soybean trypsin inhibitor
Lysozyme
MW (kDa)
102.663
79.236
46.236
33.872
27.15
17.434
The units “g/mol” and
“Dalton” are used inter-
changeably for molecular
weight in this manual.
For example, a 20,000
Dalton protein has a
molecular weight of
20,000 g/mol. A Dal-
ton is a unit equal to
1.0000 on the atomic
mass scale; very nearly
equal to the mass of a
hydrogen atom. The av-
erage amino acid residue
in a protein is 120 Dal-
tons. Thus, a protein
with a molecular weight
of 20.000 contains 167
amino acid residues
(20,000/120 = 167).
Typical Calibration Curve of Standard Proteins
1000
100
Molecular Weight (kDa)
10
y = 323.79e-0.059%
R2 = 0.9958
1
40
45
50
15
20
25
30
35
Distance Migrated (mm)
Figure 4.1:
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Experiment: 4 PROTEIN ELECTROPHORESIS; SDS-PAGE
Vertical electrophoresis apparatus, Power Supply
fisher
link to ladder
Tube
6
PART I: GEL PREPARATION
PROCEDURE:
1. Carefully remove gel cassette from wrapper.
2. Remove bottom strip of plastic. Cut with razor blade and tear carefully.
3. Gel must be loaded in the cassette apparatus in the correct orientation.
The wells of the cassette must be facing the center of the assembly.
4. Loosen screws and place cassette against the plastic structure. Next,
tighten the screws. (The object is to create a well on the inside separate
from the surrounding container.)
5. The assembly is then placed in the electrode structure. The larger plastic
tips on each side of the assembly are put in location. The small feet are
placed into the base. You must twist the footing of the assembly so the
“feet” can snap in. Watch the demonstration closely.
6. Place the completed assembly unit into the box. Remove the comb
carefully.
7. Fill in inner chamber with 1X SDS Running Buffer. Make sure the inner
chamber does not leak. The inner chamber must be filled to the top of
the walls for the connection to be made.
8. Fill the outer chamber with 1X SDS Running Buffer.
9. Run samples at about 100 mV for 5-10 minutes to allow the sample to
enter the gel. The voltage can then be increased to 120 mV, until the
loading dye reaches the bottom of the gel.
PART II: STAINING THE GEL
PROCEDURE
1. Stain with Zinc stain. Remove gels from plastic cassette carefully. The
gels are easy to tear.
2. Place gel in Stain A for 10 mins and place on shaker.
3. Remove gel and place in Stain B for 30-60 seconds. Where there is no
protein the gel will turn opaque. Where there is protein the bands will be
clear. This is called ” negative staining
4. Place in dH2O

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