Importance of “blocking” nitrocellulose membrane in Western blot method

1.In the Western blot method, it is important to “block” the
nitrocellulose membrane after electrophoretic transfer of
the proteins. Why is this the case? Choose all that apply.
Select one or more:
To minimise non-specific antibody binding
To enhance the binding of the secondary antibodyconjugate
to the primary antibody
To ensure all the sites on the nitrocellulose membrane are
occupied
To block proteases from degrading the proteins and
antibodies
2.You have used the 6His-GFP that you expressed and
purified in the lab class to generate antibodies in a rabbit.
The antiserum works in a Western blot on your protein.
What other proteins could potentially be detected in a
Western blot using this same antiserum? Select all that
apply.
Select one or more:
BSA
6His-OTC-GFP
ClpP
6His-proNaD1
OTC-GFP
3.You are working in a LIMS research lab and you are
performing a Western blot with your antiserum. However,
the lab does not have the secondary antibody that you
previously used in the prac lab. Choose a suitable
secondary antibody that is available in their fridge that
also uses the enhanced chemiluminescence (ECL)
reagent for detection.
Select one:
Rabbit anti-goat IgG-HRP conjugate
Sheep anti-rabbit IgG-AP conjugate
Mouse anti-rabbit IgG-biotin
Donkey anti-rabbit IgG-HRP conjugate
Sheep anti-goat IgG-Alexa Fluor conjugate
4.Western blot and ELISA are two antibody-based
methods that are very useful in protein biochemistry
studies. They share several similarities and differences.
Which of the following statements are not true? Select all
that apply.
Select one or more:
A given antibody should work just as well in both Western
blot and ELISA settings
Both methods can make use of a secondary antibodyenzyme
conjugate for signal amplification
Both methods can detect the specific protein of interest
out of a complex protein mixture
Some antibodies are directed to conformational/structural
epitopes that are only present in the ELISA format
ELISA requires denaturation and reduction of protein
samples prior to assaying, like in Western blots
5.The 6His-GFP protein was purified from bacterial
expression extracts using Immobilised Metal Affinity
Chromatography (IMAC). At the end of the procedure, a
Lowry protein assay was conducted to determine the
concentration of protein in the Crude Lysate (C) and
Eluant (E) fractions. For the assay, BSA was used as the
protein standard and both the crude lysate (C) and eluant
(E) samples were diluted 1/10 with water to bring them
into the range of the standard curve.
Use the assay data (HERE), to plot a protein standard
curve in Excel.
From your chart, what is the equation for the line?
y=………….x
6.Assuming the eluted sample contains a high proportion
of GFP, comment on the accuracy of the protein
estimation for this sample using the method above.
7.The variant of GFP that you have purified is EGFP
(which is able to fluoresce). It has an excitation
maximum at 488nm and an emission maximum at
509nm. This property can be exploited to determine
the specific concentration of GFP in the sample.
Sample Protein
concentratio
n in assay
Dilution
factor
Original
protein
concentratio
n in sample
Crude
Lysate
Answer
mg/ml
10 Answer
mg/ml
Eluant Answer
mg/ml
10 Answer
mg/ml
A fluorescent assay for GFP was set up using black
(opaque) microtitre plates. For the assay, highly
purified GFP was used as the protein standard and
the crude lysate (C) and eluant (E) samples were
diluted 1/25 and 1/100, respectively.
Use the assay data (HERE), to plot a protein standard
curve in Excel.
From your chart, what is the equation for the line?
y= ………. x
8.Using the GFP standard curve, calculate the protein
concentration for the crude lysate and eluant fractions and
fill in the following table.
9.The GFP was eluted in the IMAC procedure with 250
mM imidazole. There is no imidazole in the crude lysate
fraction however. It is possible that the presence of
imidazole could interfere with either of the assays. Design
a theoretical addition to this experiment that could control
for this.
Sample GFP
Concentratio
n in Assay
Dilution
Factor
Original GFP
Concentratio
n in Sample
Crude
Lysate
Answer
μg/ml
25 Answer
mg/ml
Eluant Answer
μg/ml
100 Answer
mg/ml
10.Using your responses to Questions 5 and 8, calculate
the specific concentration of GFP per mg of protein in both
your crude lysate and eluted fractions.
Specific concentration of GFP in Crude Lysate: ………..
mg GFP/mg protein
Specific concentration of GFP in Eluant: …….. mg GFP/
mg protein
What fold purification of GFP was obtained at the end of
this experiment? 8,18,20,5,15,11fold purication

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