Transcript BISC 220 Lab 2

BISC 220 Lab 2
Protein Purification
by
Affinity
Chromatography
&
Determination of
Specific Activity
TO DO TODAY
• Extract protein induced last week from the
bacterial cells
– chemical cell lysis with“Bacterial Protein
Extraction Reagent”- DETERGENT
– Removal of cell debris by centrifugation
• Partial Purification of protein
– Separate the protein of interest from other
proteins by Affinity Chromatography
• Assay the total protein in the CRUDE
EXTRACT & the PURIFIED
FRACTION and Assay the specific
activity of the b-gal in each.
• Quantify & compare total protein &
specific activity for both fractions & then
calculate % yield & purification factor
Affinity Chromatography:
the General Strategy
Specific
binding
Example:
Elution
(release)
Can use altered pH, salt,
competitor molecule for
elution.
Protein Purification via Metal
Chelate Affinity Chromatography
• The 6xHis tag on the b-gal will bind tightly
to the Ni+ agarose, which can be
separated from the supernatant by
centrifugation.
• Other proteins that interact non-specifically
(weakly) with the Ni+ agarose will be
removed during washes.
• The 6xHis b-gal will be eluted (released)
from the beads by competition with
imidazole (a molecule similar to histidine).
Imidazole & histidine: a
structural comparison
Imidazole
ring
• Excess imidazole outcompetes 6xHis-b-gal for
binding to Ni+ agarose
b-Gal Partial
Purification Protocol:
Things to Remember
• Follow directions carefully
• Know where you are in process
• Mix well, measure carefully &
don’t confuse reagents
• Be careful making dilutions
• End up with 2 fractions to assay:
– Crude Extract-CE
– Purified (partially) Fractioncontains b-galactosidase-PF
Determining Total Protein
Content Spectrophotometrically
(both CE and PF)
Free Coomassie
Blue Dye
(Bradford Reagent)
Absorbance at 470 nm
+ Protein
Dye Bound to Protein
Absorbance at 595 nm
Absorbance
Making a Standard Curve from
Absorbance readings of known
BSA concentrations using Linear
Regression
Concentration (mg/ml)
• y =m x + b, where m is the slope & b is the yintercept. Use the equation generated by Excel to
solve for x (concentration)
• Will need to dilute a 1 mg/ml BSA stock to make
0.1, 0.2, 0.4, 0.6 & 0.8 mg/ml samples (not a
dilution series; make 200 µl of each dilution)
Measuring the Specific
Activity of b-gal
Colorless
• ONPG = artificial substrate for b-gal
• Specific activity = Vmax (maximum velocity) =
rate of appearance of product under conditions
of saturating amounts of substrate
(mmol/min/mg protein)
• Detect appearance of ONP (product) by
absorbance at 420 nm.
What does the Specific
Activity tell you?
• With greater purification of an
enzyme, total activity & % yield
will decrease but specific activity
will increase.
• Purification factor = ratio of
specific activities of PF/CE; a
higher purification factor means
that more of the protein in the
sample is the enzyme of interest.
Calculating the Concentration of
ONP from A420 Readings of
Enzyme Reactions
Beer-Lambert Law:
A
C=
e*l
C = concentration (moles/L)
A = absorbance reading at given wavelength (no
units)
e = molar extinction coefficient at given l
(M-1cm-1)
l = spectrophotometer path length (cm)
e for ONP = 4800 M-1cm-1
• Must use an amount of enzyme that produces
an amount of product (in a defined reaction
time) that gives an absorbance reading in the
reliable range for the spectrophotometer (0.11.0). Will try several dilutions of CE & PF.
Making Dilutions
V1 x C1=V2 x C2
• FOR PROTEIN ASSAY
1. BSA stock 1mg/ml
Working dilutions (want 200µl):
0.1, 0.2, 0.4, 0.6, 0.8 mg/ml
2. Purified (PF) & Crude Extract (CE)
1:5 dilution with Z buffer (want 300 µl)
• FOR ENZYME ASSAY
1. Purified Fraction
Want 250µl each of 1:100, 1:200, 1:400&
1:800 dilutions
2. Crude Extract
Want 250 µl each of 1:50, 1:100, 1:200 &
1:400 dilutions
(Use serial dilution strategy for these.)
Things to Remember about
Assays:
Protein Assay
• Make 2 reagent blanks instead of
1 since using double beam
spectrophotometer- 11 tubes
• Mix dilutions well & keep on ice
• Timing is not critical
b-Gal Assay
•
•
•
•
Keep all diluted fractions on ice
Make 2 reagent blanks instead 1
Timing IS critical!!!
You will have a lot of tubes in
your ice bucket. Be VERY careful
not to mix things up—label well.
Before You Leave
• Add glycerol to remaining purified fraction &
give to instructor to freeze
• Give 3 samples to instructor (properly
labeled—see p. 41)
• Clean up your work area
Homework
• Complete calculations & answer questions on
p. 42
• Don’t wait until the last minute to do the
calculations!
• When calculating protein concentrations, don’t
forget to account for the dilutions.
• For other calculations, follow examples on p.
48-50.