determining protein molecular weight and purification of green and blue florescent proteins

I have to write a lab report 6 pages minimum about determining protein molecular weight and purification of green and blue florescent proteins. the lab report should be in 5 different sections, which are objective, background, results, discussion, and answer given questions. on the result section, we need to conduct scatter linear graph.I am attaching the instruction page as well as the ppt and all the results I got.
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BCHB
507/508
Laboratory Applications of
Biotechnology
Session 15
Lab 153: Polyacrylamide gel Electrophoresis:
Determining Protein Molecular Weight
Lab 999: Real Time PCR Demonstration
Lab 255: Purification & Size Determination of
Green & Blue Fluorescent Proteins
BCHB
507/508
Laboratory Applications of
Biotechnology
Lab 153: Polyacrylamide gel Electrophoresis:
Determining Protein Molecular Weight
Determination of Protein Molecular Weight
?
Direct Calculation: Must know amino acid sequence.
? Does
?
not take into account modifications.
SEC: Size Exclusion Chromatography.
? Calibrate
column with standards and measure sample
retention time.
?
PAGE:
? Does
not require expensive equipment.
? Native gel vs. SDS gel.
? Denature
and bind SDS (net negative charge on protein).
? Run on gel and compare to standards.
SDS-PAGE
?
?
?
SDS-PAGE = Sodium DodecylSulphatePolyAcrylamide Gel Electrophoresis)
SDS-PAGE separates proteins according to their size
Need to understand: SDS and PAGE meanings
SDS = Sodium DodecylSulfate
?
SDS = Sodium DodecylSulfate
anionic detergent
? denatures secondary and non–disulfide–linked tertiary
structures
? SDS confers a net negative charge to the polypeptide in
proportion to its length
?
?
Polypeptides become rods of negative charges with
equal charge per unit length.
Polyacrylamide Gels
?
?
?
?
Have smaller pores than agarose, therefore high
degree of resolving power
Polyacrylamide gel electrophoresis separates
proteins based on Size (Molecular Weight)
The gel (matrix) composed polyacrylamide
Acrylamide monomers polymerize into long chains
that are covalently linked by a crosslinker
? Acrylamide
is a potent neurotoxin and should be
handled with care!
Polymerization of acrylamide
TEMED – crosslinking
agent
(19:1 ratio of
acrylamide to bis
maximizes
crosslinking)
Higher % of gel smaller pores (holes)
so smaller fragments
can be resolved
http://nptel.ac.in/courses/102103047/module3/lec13/5.html
PAGE = PolyAcrylamide Gel Electrophoresis
?
?
PAGE is the preferred
method for separation
of proteins
Movement of Proteins in
Gel: smaller proteins
will move through the
gel faster while larger
proteins move at a
slower pace
http://www.biologyexams4u.com/2014_01_01_archive.html
SDS-PAGE
?
Protein sample treated with SDS
and beta-mercaptoethanol
?
?
?
SDS disrupts secondary and
tertiary structure
The mercaptoethanol reduces
disulfide bonds
Results of treatment:
All proteins contain only primary
structure and
? All proteins have a large
negative charge
?
?
They migrate through a gel
towards the positive pole at a
rate proportional to their linear
size
?
Molecular weights with respect
to size markers may then be
determined
http://www.biologyexams4u.com/2014_01_01_archive.html
Protein gel electrophoresis
Determination MW of Unknown Proteins
www.edvotek.com/153.pdf
Running SDS-PAGE
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Unsolidified gel (or Acrylamide crystals) are toxic, handle gel with gloves.
Remove gel from the pouch and remove tape.
Rinse the gel with DI water.
Carefully remove the comb and insert gel into chamber (check orientation).
Add buffer to chamber (make sure the wells are covered).
Use a transfer pipet to rinse out the wells with buffer (often there is unpolymerized material in the
well).
Boil samples for 3-4 minutes and quickly load samples into wells. (Set tip slightly into well and
expel sample.)
Set cover on gel box and electrophoresis at 125 volts for 60 minutes (watch for the dye front to
reach the bottom of the gel).
Turn off the power supply, drain and rinse the gel box and remove the gel cassette from the gel
box.
Set the gel cassette down with the smaller plate up. Using a spatula crack the edges of the
cassette to separate the top and bottom plates. Gently lift and separate the smaller plate from
the larger plate, the gel should remain on the larger plate. CAUTION: The gel is very thin and can
tear.
Prepare the tray with the fixing solution and set the plate with the gel into the solution.
Transfer the gel to a tray of staining solution and float a sheet of gel Stain in the solution.
Allow the gel to stain for 1-3 hours (if required staining can proceed overnight).
BCHB
507/508
Laboratory Applications of
Biotechnology
Lab 255: Purification & Size Determination of
Green & Blue Fluorescent Proteins
What are Green and Fluorescence
Proteins and Why study them?
?
?
?
Discovered from Aequorea
Victoria
Widely expressed in E. coli
Visual markers (“reporter
molecule”) for various studies
?
?
?
?
?
Localization and regulation of
gene expression
Cell movement
Protein-protein interactions
Screenable marker to identify
transgenic organisms
Several variants: blue, red,
ect. fluorescent proteins
https://en.wikipedia.org/wiki/Green_fluorescent_protein
GFP derivatives
Column Chromatography
?
Chromatography used
for protein purification
? Size
exclusion
? Affinity
? Ion exchange (Lab
302)
? Hydrophobic
interaction (today)
cellularphysiology.wikispaces.com
Hydrophobic Interaction Chromatography
(HIC) = Salting Out
?
?
?
Add bacterial lysate
to column matrix in
high salt buffer
Wash less hydrophobic
from column with low
salt buffer
Elute GFP from column
by adding a no-salt
buffer
Column Chromatography
?
Packing and Equilibrating the column
•
•
•
?
Collecting Column Fractions of (gfp) Proteins
?
?
?
?
?
Label eight tubes # 1-8
Load the column with gfp extract
Add 1X Elution buffer to column & collect 0.5 ml fractions in
labeled tubes
Store on ice
Collecting Column Fractions of (bfp) Proteins
?
?
?
?
?
?
Mount column on ring stand STRAIGHT.
Mix and load matrix (avoid bubbles).
Wash packed column.
Wash column
Label eight tubes # 9-16
Load the column with gfp extract
Add 1X Elution buffer to column & collect 0.5 ml fractions in
labeled tubes
Store on ice
Identifying the tubes with the highest fluorescence of
(gfp) and (bfp) using long wave U.V. light
www.edvotek.com/302.pdf
HIC vs. SDS-PAGE
SDS-PAGE
?
?
?
?
An analytical technique used
to detect the presence of a
protein of interest
Separates molecules based on
size
HIC
?
?
Can compare denatured and
intact proteins to study protein
structure
?
High concentration of GFP
from whole cell lysate samples
(dense colonies)
?
Used to isolate a protein from
a complex mixture of
molecules based on its physical
and/or chemical properties
Separates molecules based on
hydrophobicity
Need to lyse open the cells to
run the soluble proteins over
the column
Very dilute concentration of
GFP in the HIC column
fractions
Resources
?
Packing a chromatography column
? https://youtu.be/G4jyd8L0MWE
?
Performing chromatography
? https://youtu.be/VP6Px8zTDNM
?
Performing SDS-PAGE
? https://youtu.be/eaETFKXtNRA
?
Real Time PCR animation
? https://www.youtube.com/watch?v=EaGH1eKfvC0

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