ACTIVITY 6

PROTEIN ISOLATION, PURIFICATION,

AND CHARACTERIZATION

Submitted by : April Ann Rose Naparato MLS 2H

A. Extraction of Protein from Procedure:
Rat Liver 1. Remove liver aseptically.
2. Flash-freeze liver in foil packets using
Materials: liquid nitrogen. Store at –80°C.
Aluminum Foil 3. Crush liver sample on dry ice and store in
Liquid Nitrogen a prechilled 5 ml culture tube.
Dry Ice 4. Add about 100 μg tissue (enough to
5 mL Culture Tubes almost cover round portion of a 5 ml culture
Phosphate Buffered Saline tube) to a new chilled tube.
Protease Inhibitor Cocktail (Sigma 5. Add 1 ml PBS with 10 μl protease
Cat.no. P-8340) inhibitors (Sigma Cat. no. P-8340). Chill this
Quant-iT™ Protein Quantitation Kit solution using wet ice.
(Cat. no. Q33210) 6. Homogenize at low speed for ~20 seconds.
Make sure to keep cool and keep samples on
wet ice.
 B. Sub-Cellular Centrifugation
1. Transfer samples into 1.7 ml microcentrifuge tubes.
2. rats were sacrificed and the livers were promptly
excised, weighed and placed in a 5X volume of
homogenizing buffer at 4 jC.
3. . The homogenate was centrifuged at a low speed
(3000 X g) to separate the unbroken cells, red
blood cells, cell membranes, nuclei, etc.
4. The pellet was re-suspended in homogenizing medium
and homogenized and centrifuged at 3000 X g two
more times to quantitatively remove all
mitochondria.
5. The combined supernatants were centrifuged at 9000
X g for 15 min.
6. The pellet was saved and washed with 5 volumes of
homogenization buffer and centrifuged at 13,000 X g for
10 min.
7. This pellet was again washed with homogenization
buffer and centrifuged at 3000 g for 5 min to remove
contaminants as before.
8. This supernatant was centrifuged at 13,000 x g for 20
min. The final pellet was re-suspended in 2 –3 volumes of
0.25 M sucrose, pH 7.4.
 C. Ammonium Sulfate fractionation (assume
maximum enzyme activity is found in the 35-
45% ammonium sulfate fraction)
- use 40% AMMONIUM SULFATE (NH4)2 SO4 to remove AMMONIUM SULFATE and concentrate
the protein.“We choose 40% (NH4)2 SO4, for it is the average percentage from the range
given.”
D. Fast Protein Liquid Chromatography
with Gel Filtration
- Since we already have varieties of proteins from the cytoplasmic enzyme of
the rat’s liver, we can use the gel filtration with polyacrylamide beads to
separate the different sizes of protein.
E. FPLC with Affinity
Chromatography
1) Inject a sample into an initially
equilibrated affinity chromatography
column(AFpak).
2) Only the substances with affinity for the
ligand are retained in the column.
3) Other substances with no affinity for the
ligand are eluted from the column.
4) The substances retained in the column
can be eluted from the column by changing
pH or salt or organic solvent concentration
of the eluent.
 F. SDS-Page
Materials and Reagents
1. Pre-stain Protein MW marker PROCEDURE IN MAKING SDS-PAGE GEL
2. TEMED 1. Clean and completely dry glass plates, combs, and
3. Ammonium persulfate spacers are required.
4. SDS 2. Assemble gel cassette by following manufacturer
5. 30% Acrylamide stock (37.5: 1 acrylamide: instructions.
bisacrylamide)
3. Prepare 10% lower gel (separating gel) by adding the
6. Bromophenol Blue
7. Tris Base following solutions : 2 ml ddH2O,1.67 ml 30%
8. Glycine acrylamide/Bis, 1.25 ml 1.5 M Tris (pH 8.8), 25 μl 20% SDS, 25
9. EDTA μl 10% ammonium persulfate 2.5 μl TEMED
10. Glycerol 4. To avoid polymerization, after adding TEMED,mix well and
11. Isopropanol
quickly transfer the gel solution by using1 ml pipette to
12. Tris-HCl (pH 6.8)
13. β-mercaptoethanol
the casting chamber between the glass plates and fill up
14. 10x running buffer to about 0.7 cm below the bottom of comb when the comb is
15. 2x SDS protein sample buffer in place.
 F. SDS-Page
5. Add a small layer of isopropanol to the top of the gel 9. Allow the top portion to solidify and then carefully
prior to polymerization to straighten the level of the gel. remove the comb.
6. Once the gel has polymerized, start to prepare Note: The gels can be stored with the combs in place
stacking gel (5%) by adding the following solutions: 2.088 tightly wrapped in plastic wrap and put in a second
ml dH2O ,0.506 ml 30% acrylamide/Bis, 0.375 ml 1 M Tris (pH container with wet tissue towel (keep the gels moist) at
6.8), 15 μl 20% (w/v) SDS, 15 μl 10% ammonium persulfate, 4 ° C for 1 to 2 weeks.
1.5 μl TEMED
7. Remove the isopropanol layer by using filter paper. Rinse
the top layer of the gel with ddH2O and dry off as much of
the water as possible by using filter paper.
8. Add TEMED and mix the stacking gel solution well. Quickly
transfer the gel solution by using a 1 ml pipette till the
space is full, and then insert the appropriate comb.

F. SDS-Page
SAMPLE PREPARATION
1. Prepare same amount of protein samples
according to BCA assay result, see BCA
(bicinchoninic acid) protein assay.
2. Add the same volume of 2x protein sample
buffer to each protein sample, mix and boil the
samples at 95 °C heating block module for 10 min.
3. Spin the samples at the maximal speed for 1 min
in tabletop centrifuge and leave the samples at
room temperature until you are ready to load onto
the gel.
Note: Can store extracted protein samples
(containing
sample buffer) at -20 °C and reheat at 95 °C for 5
min when used the following time.
Electrophoresis
1. Remove the gel cassette from the casting
stand and place it in the electrode assembly
with the short plate on the inside. Press
down on the electrode assembly while
clamping the frame to secure the electrode
assembly and put the clamping frame into the
electrophoresis tank.
2. Pour some 1x electrophoresis running
buffer into the opening of the casting frame
between the gel cassettes. Add enough
buffer to fill the wells of the gel. Fill the
region outside of the frame with 1x running
buffer.
3. Slowly load the same amount of protein
samples into each well as well as load 10 μl
of protein MW marker.
 G. Structure of the Enzyme

Primary
Structure
Secondary
Structure
Tertiary
Structure
Quaternary
Structure
 2. Schematic Diagram
Sub-cellular Ammonium Sulfate
Extraction from rat liver
centrifugation Fractionation

FPLC with affinity FPLC with Gel

SDS-PAGE
Chromatography Filtration

Determine the primary, secondary,

tertiary and quaternary structure of the
enzyme
THANK YOU!