Transcript No Slide Title

Students: Nancy Doremus and Jessie Dodge-Gittins
Mentors: Zeb Jones and Mike Stoops
• The way a protein is assembled
and folded is very important in
determining its purpose. It’s like a
second genetic code.
• RXRalpha is a receptor protein for
retenoid (vitamin A derivatives)
molecules.
• These receptors are implicated in
cell differentiation, mammalian
development, and skin diseases like
acne and psoriasis.
Jessie weighing buffer materials
•Understand the stability of RXRalpha
•Comprehend dynamics of the protein unfolding and folding
•Fit data to a theoretical folding model
Jessie and Nancy getting ice
• Purifying the RXRalpha protein
• Checking for contamination
• Testing concentration
• Collecting data after exposure to a chemical denaturant
Jessie using the pipette
Nancy filling test tubes
The RXRalpha sample needed a higher concentration for the
experiment, so the centrifuge was used to purify it. The
centrifuge spun the protein at 4000 RPM’s for 40 minutes,
which separated the protein from the filtrate. After testing the
concentration of the protein, it was found that it wasn’t high
enough, so the centrifuge was needed again.
Looking down into the centrifuge
Nancy and Jessie loading the centrifuge
The spectrophotometer
measures the absorbency of
light through a sample.
It was used to find the
concentration of the
RXRalpha protein.
Samples of known concentration ( Bovine )
Electrophoresis gives a rough
molecular weight range. SDS (
Sodium dodecyl sulfate ) in the
gel acts as a surfactant.
Surfactant denatures proteins
allowing for band resolution as
proteins migrate through an
electric field. Multiple bands
suggest contamination.
The fluorometer shines light through a sample
which makes some amino acids fluoresce. The
machine detects this and sends the data to a
computer where it is compiled into a graph.
A denaturant ( guanadine )
was added to the RXRalpha
sample to divide the tetramers
into monomers. This change
is visible on the graphs.
• Import data into Excel
• Calculate net fluorescence (subtracting buffer blank)
• Calculate weighted mean wavelength < λ > :
<λ>=Σ(I*λ)/Σ(I)
λ = wavelength
I = net fluorescence
• Correlate calculated < λ > to known values.
Weighted-mean average wavelengths correspond well
with previous data. Each protein configuration has a
concise average wavelength, distinguishable by several
nanometers. The unfolding scheme appears to follow a
progression from tetramer to dimer and finally, as the
chemical denaturant (guanadine) concentration
approached 5.5 M, monomer. Further experiments
might assay the refolding of the protein as guanadine is
diluted.
Electrophoresis Standards
1
2
3
4
5
6
7
8
MW (daltons)
Myosin
198,000
B-galactosidase
115,000
Bovine serum albumin
93,000
Ovalbumin
49,800
Carbonic anhydrase
35,800
Soybean trypsin inhibitor
29,200
Lysozyme
21,300
Aprotinin
6,400