Transcript Protein Work

Protein Work
2004, 9, 4
Koo Na Youn
Protein Work
 Cell and tissue homogenization
 Lysis buffer
 Cell Lysis
 Protein concentration determination
 Immunoprecipitation
 SDS-PAGE & Western blotting
Cell and tissue homogenization
• Base Ingredients
Lysis Buffer
– Tris-HCl (buffering agent prevents protein denaturation)
– NaCl (prevents non-specific protein aggregation)
– NP-40 (non-ionic detergent; 10% stock solution in H20)
– Na-deoxycholate (ionic detergent; 10% stock solution in H2O; protect
from light)
• Protease Inhibitors
– Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in
isopropanol; store at room temperature)
– EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4)
– Leupeptin (store frozen in aliquots, 1 mg/ml in H2O)
– Aprotinin (store frozen in aliquots, 1 mg/ml in H2O)
– Pepstatin (store frozen in aliquots, 1 mg/ml in methanol)
• Phosphatase Inhibitors
– Activated Na3VO4 (200 mM stock solution in H2O; Activation of Sodium
Orthovanadate)
– NaF (200 mM stock solution; store at room temperature)
Activation of Sodium Orthovanadate
Lysis Buffer
1. Prepare a 200 mM solution of Na3VO4.
2. Adjust the pH to 10.0 using either 1 N NaOH or 1 N HCl. At
pH 10.0 the solution will be yellow.
3. Boil the solution until it turns colorless (approximately 10
minutes).
4. Cool to room temperature.
5. Readjust the pH to 10.0 and repeat steps 2, 3 and 4 until the
solution remains colorless and the pH stabilizes at 10.0.
6. Store the activated sodium orthovanadate as aliquots at -20℃.
Reference: Gordon, J., Methods Enzymol. 201: 477-482, 1991
Lysis Buffer
Preparation of Modified
Radioimmunoprecipitation (RIPA) Buffer
The final concentrations in the modified RIPA buffer should be:
–
–
–
–
–
–
–
–
–
Tris-HCl : 50 mM, pH 7.4
NP-40 : 1%
Na-deoxycholate : 0.25%
NaCl : 150 mM
EDTA : 1 mM
PMSF : 1 mM
Aprotinin, leupeptin, pepstatin: 1 μg/ml each
Na3VO4 : 1 mM
NaF : 1 mM
Store RIPA buffer at 2-8℃ until ready to use.
Preparation of Cell Lysate
Cell Lysis
1. Wash adherent cells twice with ice-cold PBS. Wash nonadherent cells in PBS and centrifuge at 800 to 1000 rpm in a
table-top centrifuge for 5 minutes to pellet the cells.
2. Add ice-cold modified RIPA buffer to cells
3. Scrape adherent cells. Transfer the cell suspension into a
centrifuge tube. Gently rock the suspension on either a
rocker or an orbital shaker in the cold room for 15 minutes to
lyse cells.
4. Centrifuge the lysate at 14,000 x g in a precooled centrifuge
for 15 minutes. Immediately transfer the supernatant to a
fresh centrifuge tube and discard the pellet.
5. Dilute the cell lysate at least 1 : 10 before determining the
protein concentration
Protein Concentration Determination
•
Protein-dye conjugate: Absorbs light
differently than the dye alone
•
Quantified: spectrophotometer
1. Bradford
• Coomassie Blue G-250
2. Lowry
3. Bicinchoninic Acid (BCA)
Protein Concentration Determination
Bradford Assay
• The change in absorbance in Coomassie
Blue G-250 upon binding of protein
• Not susceptible to interference by a wide
variety of chemicals present in samples.
• The notable exception is high concentrations
of detergents.
Protein Concentration Determination
Lowry & Bicinchoninic Acid (BCA)
• BCA reduces divalent copper ion to the
monovalent ion under alkaline
conditions, as is accomplished by the
Folin reagent in the Lowry assay.
• The advantage of BCA is that the
reagent is fairly stable under alkaline
condition, and can be included in the
copper solution to allow a one step
procedure.
• A molybdenum/tungsten blue product is
produced as with the Lowry.
Procedure
Bradford Assay
Note: do all determinations in duplicate or triplicate
1. Pipette 0, 2, 4, 6, 10, 15 and 20 µl of BSA (1 mg/ml) into
assigned wells of a 96-well plate.
2. Pipette up to 20 µl of unknown samples into individual wells
of a 96-well plate.
3. Add 40 μl of Bradford Reagent into all wells containing
standard or sample.
4. Add dd H 2O to all wells to bring the final volume to 200 µl.
5. Read absorbance at 595 nm without any prior incubation.
Immunoprecipitation
Immunoprecipitation
Protein A/G Agarose bead
Specific Antibody
Cell
extracts →
A/G
A/G
A/G
A/G
Discard the supernatant fraction
wash the
beads 3-5
times
Centrifuge
1000rpm, 3min
4℃,
4hr or O/N
Agitation
Resuspend the
agarose/sepharose
beads in 2 x sample
buffer and mix gently.
A/G
Immunoprecipitation
1. Prepare cell lysate
2. Prepare protein A or G agarose/sepharose
3. Pre-clear the cell lysate by protein A or G
agarose/sepharose bead
4. Determine the protein concentration of the
cell lysate
Immunoprecipitation
2. Prepare protein A or G agarose/sepharose
1.Wash the beads twice with PBS
2.Restore to a 50% slurry with PBS.
3.It is recommended to cut the tip off of the pipette tip when
manipulating agarose beads to avoid disruption of the
beads.
Immunoprecipitation
3. Pre-clear the cell lysate by protein A or G
agarose/sepharose bead
1.Pre-clear the cell lysate by adding 100 microliters of
either protein A or G agarose/sepharose bead slurry
(50%) per 1 ml of cell lysate and incubating at 4℃ for 10
minutes on a rocker or orbital shaker.
2.Remove the protein A or G beads by centrifugation at
14.000 xg at 4℃ for 10 minutes. Transfer the supernatant
to a fresh centrifuge tube.
Immunoprecipitation
4. Determine the protein concentration of the
cell lysate (Bradford assay)
1.Dilute the cell lysate at least 1:10 before determining the
protein concentration because of the interference of the
detergents in the lysis buffer with the Coomassie-based
reagent.
2.Dilute the cell lysate to approximately 1mg/ml total cell
protein with PBS to reduce the concentration of the
detergents in the buffer.
3.A more concentrated cell lysate (i.e., 10 mg/ml) may be
necessary to immunoprecipitate a protein which is found
in low levels in a cell model.
Immunoprecipitation
Procedure of Immunoprecipitation
1.Add the recommended volume of the immunoprecipitating
antibody to 500 microliters of cell lysate.
2.Gently mix the cell lysate/antibody mixture for either 2 hours
or overnight at 4℃ on a rocker or an orbital shaker.
3.Capture the immunocomplex by adding 100 microliters
protein A or G agarose/sepharose bead slurry and gently
rocking on either a rocker or orbital shaker for either 1 hour
or overnight at 4℃.
4.Collect the agarose/sepharose beads by pulse centrifugation
(5 seconds in the microcentrifuge at 14,000 rpm). Discard
the supernatant fraction and wash the beads 3 times with
800 microliters ice-cold modified RIPA buffer.
5.Resuspend the beads in 2 x sample buffer
6.The agarose/sepharose beads are boiled for 5 minutes to
dissociate the immunocomplexes from the beads.
Immunoprecipitation
Table 1. Relative Affinity of Immobilized Protein A and Protein G for Various
Antibody Species and Subclasses of Polyclonal and Monoclonal IgG’s.
Protein A: Fc portion of IgG
Protein G : Fab and Fc portion of IgG
SDS-PAGE &Western Blot
• Sodium Dodecyl Sulfate - Polyacrylaminde
Gel Electrophoresis (SDS-PAGE)
– Gel: a cross-linked matrix
• Western Blot
– Western blot analysis can detect one
protein in a mixture of any number of
proteins while giving you information about
the size of the protein.
SDS-PAGE
Western Blot
Choice of Membrane
• Nitro Cellulose (NC)
– Choose By Detection Systems : Radiolabeled,
Cromogenic and Chemiluminescent Detection Systems
– Choose By Procedures : Westerns, Protein &
Immunoblotting, Northerns, Southerns
• Polyvinylidene fluoride (PVDF) membrane
– Benefits : Hydrophobic PVDF membrane is designed for
protein sequencing,
– Choose By Detection Systems : Chemical compatibility
allows the use of all commonly used stains
– Choose By Procedures : Western, Transfers, Protein
Sequencing, Amino Acid Analysis
Western blot detection methods
1.
Chemiluminescent: Alkaline phosphatase (AP),
Horseradish peroxidase (HRP)
2.
Bioluminescent: luciferin-base derivative,PVDF
3.
Chemifluorescent: Fluorogenic compound
4.
Fluorescent: FITC, Texas Red, rhodamine
5.
Autoradiography: Rodioactivity
6.
Colorimetic
•
AP: 2-bromo-4-chloro-3-indolyl
phosphate/Nitroblue Tetrzolium (BCIP/NBT)
•
HRP: 4-chloro-1-naphthol (4CN)
•
Densitometer
Western blot detection methods
Table 2. Comparison of western blot detection methods
Luminescent
(Chemiand Bio-)
Chemifluor Fluorosescent
cent
Radioisotopic
Colorimetric
Sensitivity
Excellent
Very good
Very good
Excellent
Very good
Economy of
antibody use
Excellent
Very good
Excellent
Excellent
Good
Speed of
detection
Excellent
Excellent
Excellent
Poor
Good
Ease of
reprobing
Very good
Fair
Fair
Fair
Poor
Ease of
quantitation
Very good
Fair
Excellent
Excellent
Fair
Durability of
results
Excellent
Fair
Excellent
Excellent
Good
The End