Experiment 7 – Lab Report
Western Blot Analysis of rGFP fractions
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1.
(10pts) You will receive these points if you performed the SDS-PAGE and the Western
Transfer correctly.
2.
(2pts) What is the GENERAL purpose of a Western Blot?
Detect presence of rGFP by using a specific antibody
3.
(2pts) Ponceau S is a red dye that reversibly binds to all proteins. We used this dye to stain
the membrane prior to developing the Western blot with antibodies.
One student’s blot did
not have any bands after performing the Ponceau S stain.
Describe one likely
procedural
error during the transfer set-up
that would account for this observation.
The protein side of the membrane was facing down instead of upside and so the
dye did not bind to it
4.
(2pts) Why did we use a primary antibody that recognizes the Xpress epitope tag and not the
His
6
tag to detect rGFP?
The his6 tag could have bound other contaminants besides rGFP but
xpress epitope tag is more specific for the antibody
5.
(5pts) Make a figure of your Western blot result with appropriate titles and labels.
Please see page 7-4
6.
(2pts) Describe the data from the G0, G3, and GCE lanes of your Western blot result. Are
those results expected? Explain why or why not.
G0 had no bands. G3 and GCE lanes had bands in them. These results were
expected because at G0 there was no IPTG while G3 was induced with
IPTG and rGFP was expressed
7.
Based on your knowledge of Experiments 3-7, when you developed the Western blot:
a.
(1pt) What MW band in your elution lanes are you expecting to observe in the final
result?
About 32 kD
7-
2
