L6 Stud S17 SDS

The Big Picture
Experiment 6 SDS-PAGE/Coomassie Blue Analysis

of rGFP Fractions
1. Electrophoresis SDS-PAGE theory and gel set-up
2. How to determine Protein purity, MW, and Yield
3. Specific activity and purity

Hes sir
BIOL3380-Spring 2017
Experiments #3-8
The overall goal/purpose of theses series of experiments is

to see if we can express and purify a His6-tagged
recombinant form of GFP (rGFP) from the E. coli strain
BL21(DE3)<pLysS><pRSETA-GFPUV> using the Ni+2
agarose affinity chromatography technology.
This weeks experiment will use protein separation and
visualization to determine:
Combining this data with results from the Bradford assay

will allow us to determine:
Express rGFP in E. coli
Grow the E. coli and induce (turn on) the expression (transcription/translation) of rGFP
Harvest
Grow 3 hours 15ml centrifuge decant
supernatant
G0 G3 G3-15ml
Sample Sample pellet
Induce
expression
with IPTG
G0 = G culture before induction

G3 = G culture 3 hours post induction
Experiment #4 - Purification of rGFP using Ni+2 agarose
GCE
isolate a purify His6
crude extract tagged proteins Develop column with
a wash buffer followed
freeze/thaw Ni+2 affinity by an elution buffer
G3-15ml method chromatography
pellet
GCE
GCE = rGFP Crude Extract

W = wash fraction Collect and store samples for further analysis
E = elution fraction E1
W1
E1 little to no
fluorescence
W2/3 slight E2/3 strong

fluorescence fluorescence
W6
E6
Washes Elutions
Experiment #5
Determining Protein concentration of rGFP fractions
Abs (595nm)
W1-W6
protein (ug)
Bradford Create a BSA standard curve
Assay Using a standard curve we can
E1-E6 estimate the total protein amount
in a sample by interpolation
off the standard curve.
Knowing the volume of the

sample we can calculate the
protein concentration
Experiment #6
SDS-PAGE/Coomassie Blue analysis of rGFP fractions
Purpose
Experiment #7/8
SDS-PAGE/Western Transfer and Western Blot
G0
Purpose
Confirm the presence of rGFP
G3
using specific antibodies
GCE
probe and
transfer to
detect
nitrocellulose
W2-W3*
E2-E3*
Electrophoresis: molecules are separated in an electric
field.
Rate of migration depends upon:

1) Electrical field strength
2) of the particle
Electrode Electrode
Sample
Rate of migration depends upon:
3) Viscosity of electrophoretic medium
Paper cellulose polymer
Agarose gel carbohydrate polymer (seaweed)
Polyacrylamide synthetic polymer of

acrylamide and bis-acrylamide
4) Shape of molecule
DNA +
5) Interaction of molecule of interest with other DNA protein
molecules in the electrophoresis system
SDS-PAGE gel electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SDS is an
Used to estimate the relative amount of a particular protein

in a mixture of proteins
is normally expressed as a
Used to estimate the molecular weight ( ) of a

specific protein by comparing it to a molecular weight (MW)
standard.
Other common names for the MW standards are:
Standard Standard ladder Ladder
Marker MW ladder MW standard
MW marker
Protein Detection using Coomassie Blue Stain
Before Staining
___%Stacker
___% Resolver dye front
artifact
6
5
4 (might appear as faded doublet)
2 Our MW ladder contains 6 bands. Molecular

1 weights of the bands are not shown here.
acrylamide acrylamide polymer
monomer
APS
TEMED
acrylamide : bisacrylamide
29 : 1 ratio
APS
+
TEMED
SDS = sodium dodecyl sulfate
tail charged
Head group
Protein before adding

Charged R-groups
Hydrophobic areas
Protein after adding
Protein complex with disulfide bridge before adding reducing agent
H
Disulfide Bridge Reducing agent like
DTT or 2-ME H
Protein subunits after reducing agent
DTT = dithiothreitol
2-ME or b-ME = 2-mercaptoethanol
Effect of acrylamide percentage in the
resolver gel on protein mobility
Note: Identical
protein mixtures of
known molecular
weights (kDa)
electrophoresed
under the same
voltage for the same
BIOL3380-Spring 2017 Resolver Resolver Resolver amount of time.
Estimating ______ & ___________of a Specific Protein
Lane 1 2
150 kD
100 kD
75 kD
50 kD
25 kD
The molecular weight of a protein can be commonly estimated using one
of three methods:
Calculated molecular weight if you know the gene sequence then you can
translate that sequence into an amino acid sequence. We know the molecular
weights of each type of amino acid so we can calculate the molecular weight
of a protein.
Estimated molecular weight If you know the number of amino acids in a

protein (but not the specific sequence) then you can estimate the total
molecular weight if you know that an average amino acid weighs
approximately 120 Daltons.
Relative molecular weight you can run an SDS-PAGE and compare the
migration of a specific protein with respect to the migration of a set of
standards (a mixture of proteins in which you know their molecular weights).
A rough comparison can be performed visually. A more accurate comparison
can be performed by measuring distances traveled an plotting a standard
curve.
Estimating ______of a Specific Protein
Lane 1 2 Lane 1 = ladder

Lane 2 = sample
Bradford data = 3ug/50ul
150 kD
100 kD
A
75 kD
B
50 kD
C
25 kD
Proteins migrate at the log of their molecular weight
Creating a Standard Curve using semi-log paper and a ruler
150kD 100kD
cm cm
molecular weight (kD) 200
100
50
10
distance traveled (cm)
Heat Heat
SDS SDS
2-ME
100 kDa
25 kDa
SDS-PAGE Lab Attendance
The Week in Review
Experiment #6 SDS-PAGE/Coomassie Blue Analysis of rGFP
Fractions
1. Demo on pouring a resolving gel
Pour resolving gel
2. Demo on pouring stacker gel and preparing samples

Pour stacker gel and begin preparing samples
3. Demo on setting up gel box, loading, and running gel

Set up gel box, load, run gel
4. Demo on gel removal, staining, and destaining

Remove gel, stain, destain
Coomassie blue stains skin and clothing dress appropriately

It's not too late to get ready for Exam 2.
We have several things to help you:
Exam 2 will cover Lectures 4-6 and Labs 4-5.
There is an optional exam review on Sunday March 5th from 7-9pm in SLC 1.102. Come out
and try to "Baffle the Biologist." You ask the questions and Dr. Rippel will answer them. Don't
worry if you are unable to attend, no new course material will be presented.
The written exam is on Monday March 6th from 3 pm -4:15 pm. Because of room scheduling,
the exam will end promptly. You will NOT need a calculator for this exam. Be smart, plan to
leave your cell phones off and inside your backpacks/purses. REMAIN OUTSIDE THE ROOM
UNTIL WE CALL YOU IN TO ASSIGNED SEATING. Bring your ID card!
The calculations exam will be given during your regularly scheduled lab session (March 7-10th).
You NEED to bring a calculator. NO cell phone OR programmable calculators will be allowed!
In the "Practice Exam Problems" tab of eLearning there is a handout that has example
questions that will be found on the calculations exam. Answers to this handout will be posted at
a later date.
We look forward to everyone succeeding on the exam!
Lab Report #6 is not due until the week of March 20th (after the spring break).
SDS-PAGE Exam Spring Week of Lab 7
Lab #6 Week Break Lab Report 6
due
The Be Able Tos:
Experiment #6 SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions
1. What is the main purpose behind a SDS-PAGE gel?

2. Be able to draw/diagram and correctly label a basic SDS-PAGE gel.
3. What is the underlying principle behind SDS-PAGE gel?
4. What is the purpose of the SDS in the system?
5. What is the purpose of the b-mercaptoethanol in the sample-loading
buffer?
6. What is the purpose of the glycerol in the sample-loading buffer?
7. What is the purpose of the bis-acrylamide?
The Be Able Tos:
Experiment #6 SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions
8. Knowing the purposes of the chemicals listed above, predict what might
happen if they are left out of the system.
9. Given an appropriate figure of a SDS-PAGE gel, be able to determine: the
molecular weight, the percent purity, and the estimate yield of a specific
protein.
10. Be able to construct and use a standard curve to determine the molecular
weight of a protein.
11. Design an experiment to determine if a protein is monomeric or oligomeric.
If oligomeric, does the protein consist of only one type of subunit? If
oligomeric, are they held together by disulfide bonds?

L6 Stud S17 SDS

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The Big Picture

Experiment 6 SDS-PAGE/Coomassie Blue Analysis

2. How to determine Protein purity, MW, and Yield

3. Specific activity and purity

The overall goal/purpose of theses series of experiments is

Combining this data with results from the Bradford assay

G0 = G culture before induction

GCE = rGFP Crude Extract

W2/3 slight E2/3 strong

Knowing the volume of the

Rate of migration depends upon:

Agarose gel carbohydrate polymer (seaweed)

Polyacrylamide synthetic polymer of

Used to estimate the relative amount of a particular protein

Used to estimate the molecular weight ( ) of a

___% Resolver dye front

2 Our MW ladder contains 6 bands. Molecular

Protein before adding

Protein after adding

Protein subunits after reducing agent

Estimated molecular weight If you know the number of amino acids in a

Lane 1 2 Lane 1 = ladder

2. Demo on pouring stacker gel and preparing samples

3. Demo on setting up gel box, loading, and running gel

4. Demo on gel removal, staining, and destaining

Coomassie blue stains skin and clothing dress appropriately

Exam 2 will cover Lectures 4-6 and Labs 4-5.

We look forward to everyone succeeding on the exam!

1. What is the main purpose behind a SDS-PAGE gel?

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