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 Biochemistry Lab
Western blot Part 2 – Developing the blot General Instructions
Background
Immunoblotting (western blots) uses specific
interactions of antibodies to detect a protein of
interest. Western blotting can be divided into
two steps: transfer of the protein from the gel
to the matrix (paper) and detection of the
epitope with antibodies. Last class we
transblotted the SDS-PAGE gel onto the PVDF
membrane (transfer paper) and left the blot in
blocking solution. We will now finish the
second half of the immunoblot, the detection.
color. The product of the alkaline
phosphatase with the BCP/NBD is insoluble
and will permanently precipitate onto the
paper. This type of detection gives very sharp
bands with low background staining of the
membrane. This is one of the easiest methods
for western blot detection.
There are two disadvantages to this method:
the signal or bands fade over time when
exposed to light, and second, it is not as
sensitive as the some of the other
methods such as chemiluminesence.
There are several methods to
develop a western blot
including radioactivity,
The steps are listed below. Blocking
chemiluminescence,
with dry milk covers un-reacted
fluorescence and colormetric
portions of the blotted paper.
systems. Each of these
Remember the only protein on the paper
methods uses a series of
is from the gel. If we were to add the
incubations and washes to
antibodies directly to the paper most of
allow specific antibodies to
the primary antibody will non-specifically
react with the antigens or
bind to the paper. The blot is then
target proteins. The antibody
washed off with salt and detergent
that recognizes the protein on
(tween 20) which is essential
the paper is
in washing to eliminate
considered the
Enzymatic detection of membrane-­‐bound antigens. overall non-specific
primary antibody. A
1. Dry milk blocks the unoccupied sites on the hydrophobic interactions. At
second antibody,
membrane. 2. Primary antibody to a specific antigen is 0.5%, tween 20 will not
incubated with the membrane. 3. An antibody enzyme known as the
disrupt binding of primary
conjugate is added to bind to the primary antibody. 4. secondary antibody,
Color d
evelopment r
eagent i
s t
hen a
dded t
o t
he b
lot. antibodies to antigens, but
is then used to detect
The HRP or AP enzyme catalyzes the formation of a will optimize detection by
the primary. This
colored precipitate from the substrate at the site of the eliminating the non-specific
secondary antibody is
antigen-­‐antibody complex. interactions. Inclusion of the
often linked to an
blotting solution will help reduce non-specific
enzyme that will allow for the detection of a
interactions with proteins from the gel and the
protein. Chemiluminecent techniques use
antibody (nothing works perfectly). The
secondary antibodies conjugated (linked) to
primaries are washed away and secondary
horseradish peroxidase to generate light
antibody added. Again the blotting buffer is
wherever the complex of antigen, primary and
included to reduce background and nonsecondary proteins/antibodies are located.
specific signals. The secondary antibodies are
This signal is detected by exposing the blot to
washed away and color substrate is added to
x-ray film.
develop the blot.
We will be using secondary antibodies
TTBS / PBST – Tris/PBS buffered saline
conjugated to alkaline phosphatase. This
solution with Tween 20: A buffered (Tris or
enzyme will metabolize the substrate
PBS) solution with salt and detergent to limit
(bromochloroindoyl phosphate/nitro blue
weak, non-specific protein interactions.
tetrazolium, or BCIP/NBT) to give a blue-black
Biochemistry
Lab
Western blot – Part 2 Western Blot Procedure:
Buffers: PBST or TTBS: as needed for washing. Blocking Buffer: 20 ml of blocking buffer (see last expt for
description) to use in 1o and 2o antibody.
1. Wash the blot.
•
Pour out old blocking solution and rinse with 5-10 ml of TTBS/PBST.
•
Incubate at room temp with shaking for 2 min.
2. Incubate with primary antibody.
•
Decant TTBS/PBST
•
Add in 5 ml of 1:1000 primary antibody in diluted blocking buffer
•
Incubate at room temp with rocking for 45-60 min.
3. Wash the blot 3 times.
•
Pour out old solution and rinse with 10-20 ml of TTBS/PBST.
•
Incubate at room temp with shaking for 3-5 min.
•
Repeat last two steps a total of three times.
4. Incubate with secondary antibody.
•
Pour out TTBS.
•
Add in 10 ml of 1:10,000 secondary antibody diluted in blocking buffer.
•
Incubate at room temp with shaking for 30 min.
5. Wash the blot 3 times.
•
Pour out old solution and rinse with 20 ml of TTBS/PBST.
•
Incubate at room temp with shaking for 5 min.
•
Repeat last two steps a total of three times.
6. Develop Blot (do this step just before needed).
•
Pour out old solution.
•
Add 100 µl of BCIP and 100 µl NBT to 10 ml of color developing buffer (Note: the stock color
developing buffer is 25X).
•
Incubate with shaking at room temp until color develops.
7. Wash with water and let dry on test tube rack in drawer.
8. Take picture of blot results with camera.
Biochemistry
Lab
Western blot – Part 2 SDS PAGE – Western Blot Homework (25 pts):
Answer each of the following bullets for the homework assignment.
•
•
•
Following the gel electrophoresis and coomassie blue staining, take a picture of the gel. Also take an
image of your Western blot.
Create publication quality figures of both the gel and the blot WITH figure legends (two separate
figures). Label both the SDS PAGE and Western blot as shown in the SDS PAGE instructions from
part one. If you are unsure on how to create a publication quality image, please refer to the lab
notebook/lab math handout AND/OR use an example from the Journal of Biological Chemistry
(www.JBC.org).
Measure the distance of migration of the proteins as well as that of the tracking dye (bromophenol
blue). Distance of migration is measured from the beginning of the resolving gel to the center mass of
a protein band.
o Calculate the Rf values for each of the protein standards and the purified protein using the
formula below:
Rf = Distance of protein migration / distance of tracking dye migration (edge of gel)
o
o
Plot the log of the known protein molecular weights as a function of the Rf. The area in the
middle of the gel should yield a straight line.
Interpolate the molecular weight of the protein from the graph
IMPORTANT POINT – CRITICAL THINKING!
How do you know which band on the gel is your protein? How can you identify the protein as your purified
protein? Just having a band or the biggest, thickest SDS-PAGE band doesn’t mean it is the protein you are
interested in. Does your Rf calculated molecular weight match that which you calculated from the amino acid
sequence? How does this relate to the Western blot?
•
Compare the gel vs. the blot. Use the following points to write up a description of your results: Does
the band you believe is your pure protein stained in the SDS-PAGE gel correlate to the band identified
by the Western blot? Based on the gel, how pure is the preparation? Are there other protein bands
that are found in the Western blot? If there are, why are those bands in the blot present? How does
the molecular weight of your purified protein, as determined by the SDS-PAGE gel, match the results
determined by the amino acid sequence? If the two values are different, why? Don't assume the
differences are mistakes or one is better than another. Instead think of the different conditions that
each measurement was taken under and relate that to the differences in results.