Practical 2: Cellular Protein Localisation Using SDS-PAGE &

Western Blotting - Day 2

Introduction
In last week’s practical we separated out proteins from two human cancer cell line lysate fractions using SDS-
PAGE and transferred those separated proteins onto PVDF membrane. This week we will use an antibody to
attempt to detect one specific protein from the thousands of proteins transferred to the membrane.
Just separating proteins on SDS-PAGE gels is perfect for many applications, but what if you wanted to
determine the presence or absence of one specific protein? What are the chances that your protein of interest
will have a unique size in the human proteome? [1]

CELL BIOLOGY BY THE NUMBERS PG. 49

As you can see, there are many proteins of the same size, even in a simple E. coli proteome. As a result, we will
need to use another method to specifically detect your protein of interest. This is where Western Blotting
comes into play.
Transferring proteins out of gels and onto membranes was first described in 1979 and the name Western
Blotting followed in 1980 [2,3]. The name Western Blotting was a play on words from two previous methods
called Southern and Northern blotting which transferred DNA and RNA (respectively) out of gels and onto
membranes. Transferring the proteins out of the gel enables us to use antibodies to specifically detect the
protein of interest.
What are antibodies?
Antibodies are glycoproteins present in our bodies as part of the immune
system that detects pathogenic microbes and viruses and facilitates a
response. A large diversity of antibody protein sequence and structure is
generated by B cells and then this diversity of structures samples
proteins to detect pathogen-specific sequences, called antigens, that
trigger specific antibody production. Antibody structure consists of a
heavy and light chain encoded by different genes. Within the heavy- and
light chains are constant-regions, that are identical between different
antibodies, and variable regions, which are different for every antibody.
These variable regions are responsible for binding to antigens (antibody
WWW . JENABIOSCIENCE . COM
generating molecules) and the feature of antibodies that we are going to
harness in this experiment.
How do you make antibodies against a protein?
Generating an antibody against a protein of interest can be done in several ways. Polyclonal antibodies are
created by injecting the purified antigen into rabbits, goats, rats, or mice. The animal will mount an immune
response to the injected protein and you then sacrifice the animal and harvest its blood. The upside to
polyclonal antibodies is that you will get a lot of them and they will likely react very sensitively to your antigen
because there will be different antibodies in the mix with different epitope recognition sites. A downside is
that polyclonals tend to be less specific because there will be antibodies in the mix that react to proteins other
than your antigen of interest.

Monoclonal antibodies are generated from the combination of a B-cell producing antibodies against a given
antigen and a myeloma cancer cell. The combined cell is called a hybridoma and is immortal and produces
large quantities of one antibody that has specificity against one epitope of an antigen. These monoclonal
antibodies tend to be highly specific to the antigen and batch-to-batch variation of the produced antibody is
significantly reduced from polyclonals. A downside to monoclonal antibodies is that to make them takes a long
time and is very labour intensive.

Why not just use Western Blotting and skip the SDS-PAGE step?
It is true that we could just attach all the protein from a human cell onto a membrane and probe this with an
antibody, this technique is called ‘dot blotting’. Unfortunately with dot blots we would lose some important
information (e.g. protein size) from the measurement. Additionally, non-specific interactions between the
antibody and other proteins in the complex mixture will be missed due to all proteins being compressed
together on the dot blot.
Limitations to Western Blotting
Western Blotting also has limitations. For instance, you cannot detect a specific protein unless an antibody has
already been developed against that protein. Most proteins of interest do not have antibodies against them
since developing an antibody takes a lot of time and money and will not be invested by research groups or
companies except for well-studied proteins.

What are we doing today?

Today you will finish the experiment you started in last week’s
practical class by (1) blocking the membrane with a standard protein
to occupy protein binding sites, (2) incubating with a primary (1°)
antibody, (3) washing of any unbound 1° antibody, and (4)
incubating with a secondary (2°) antibody linked to an enzyme
(horse-radish peroxidase) that will generate visible light when we
add a suitable substrate. Demonstrators will visualize the light
emitted from your blot later in the week and post the images to
iLearn.
Part 1: Blocking
Before probing our proteins on the PVDF membrane we need to do what is called ‘blocking’. This step ensures
that all the sites on the PVDF membrane that can bind protein are occupied. This step is needed because the
antibody that will be used to probe the blot for our protein of interest is also a protein and has a high affinity
for unoccupied sites on the membrane. If we didn’t block we would see the antibody binding to not just our
protein of interest but to huge areas of the blot, making detection of our protein difficult or impossible.
Membranes are typically blocked with a homogenous protein, the two most common being the proteins in
skim milk or bovine serum albumin. In this experiment we will use 5% BSA protein in TBS-T buffer to block the
membrane.

Equipment
Rocking platform
Klipo box to hold membranes (labelled with your group number and names)

Reagents and Materials

Blocking Solution (5% BSA in TBS-T)
*** GENERAL WARNING: Do not allow the blot to dry out at any point. Always have the next solution ready
before pouring off the last of a set of washes. ***

Protocol
1. Pour off the TBS-T solution into a waste container.
2. Add 8 mL of Blocking Solution to membrane.
3. Rock for 45 min/room temp.

Part 2: Primary (1°) Antibody Incubation

Following the blocking step we have incubated your membrane with a primary monoclonal antibody raised in
rabbit against β-catenin (Cell Signalling Technologies, cat #: 8480). We will use the antibody at 1:8,000 dilution
in blocking buffer at room temperature for 1 hour.

Reagents and Materials

Blocking Solution (5% BSA in TBS-T)
1° antibody - rabbit anti-β-catenin (Cell Signalling Technologies, cat #: 8480)
*** GENERAL WARNING: Do not allow the blot to dry out at any point. Always have the next solution ready
before pouring off the last of a set of washes. ***

Protocol
1. Add 1.5 µL 1° antibody to 8 mL Blocking Solution.
2. Put cap on and mix by inverting 3 times.
3. Pour off the Blocking Solution from the membrane into the waste container.
4. Pour 1° antibody mixture onto membrane.
5. Rock for 45 min/room temp.
Part 3: Wash off unbound (1°) antibody and add secondary (2°) antibody
Reagents and Materials
TBS-T
2° antibody - goat anti-rabbit IgG-HRP (Cell Signalling Technology, cat # 7074)
Blocking Solution (5% BSA in TBS-T)
*** GENERAL WARNING: Do not allow the blot to dry out at any point. Always have the next solution ready
before pouring off the last of a set of washes. ***

Protocol
1. Pour 1° antibody solution off the membrane into waste container.
2. Add 10 mL of TBS-T to the blot and put on rocking platform for 8 min (use timer)
3. Pour off TBS-T into waste container.
4. Repeat 2 more times (3 washes total). On the last wash do not pour off the wash until you are ready with
the 2° antibody solutions in the blocking buffer (step #5 below)
5. Prepare 2° antibody solution: Add 1.5 µL of the goat anti-rabbit IgG-HRP 2° antibody to 10 mL blocking
solution. Put cap on and mix by inverting 3 times.
6. Pour off the last Wash Solution from the membrane into the waste container.
7. Pour 2° antibody mixture onto membrane.
8. Close box, ensure it is properly labelled, place into tubs at side of lab.
9. You’re done!

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THE BELOW INFO IS FOR YOUR LAB REPORT USE ONLY AND WILL BE PERFORMED BY THE DEMONSTRATORS

Part 4: Visualisation
We will now identify where on the blot the primary and secondary antibody are bound. This information will
enable us to determine if we have detected the protein we intended to and whether this antibody works. An
enzyme linked to the secondary antibody, called horseradish peroxidase, will be used to visualise where on the
blot the primary and secondary antibodies are located. We can then compare this information to the image of
total protein attached to the blot taken last class.

Equipment
Scissors
Klipo box
Flat Plastic forceps
LiCOR C-DiGit® Blot Scanner

Reagents and Materials

Plastic sleeve
Enhanced chemiluminescence (ECL) reagents A and B – 300 µL each
15 mL Falcon tube
Protocol
WASH
1. Pour off the 2° antibody solution
2. Add 10 mL of TBS-T to the blot and put on rocking platform for 8 min
3. Pour off TBS-T
4. Repeat 2 more times (3 washes total). On the last wash do not pour off the wash until you are ready with
the ECL solution.

ECL INCUBATION
5. Cut the edges off the plastic sleeve
6. Mix 300 µL of reagent A and 300 µL of reagent B from the ECL reagent kit in an eppendorf tube
7. Pick up blot with forceps and lay FACE UP onto the open plastic sleeve
8. Add the mixed ECL reagents to the top of the blot
9. Lay the top of the plastic sleeve over the blot and ECL reagent, spreading the ECL solution out even across
the blot. Make sure there are no bubbles over the blot.
10. Tell demonstrators that you’re ready to scan your blot.
11. Incubate for 5 min (set timer)
12. Remove excess liquid from the sides of the plastic sleeve with Kimwipes.
13. Demonstrators/technician will take you up in groups to E8C 330 (6WW 330) lab to visualise the blot by
scanning with the LiCOR C-DiGit® Blot Scanner.

References
1. Ron Milo and Rob Philips. Cell Biology by the Numbers. Garland Science (December 7, 2015). Pg. 48.

2. Towbin H, Staehelin T, Gordon J (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications". Proceedings of the National Academy of Sciences USA 76 (9): 4350–54. doi:10.1073/pnas.76.9.4350

3. Burnette WN. (1981). "'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to
unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A". Analytical Biochemistry 112 (2): 195–
203. doi:10.1016/0003-2697(81)90281-5