Lovely

PROFESSIONAL
UNIVERSITY
TERM PAPER
OF
CONCEPT OF BIOTECHNOLOGY

TOPIC:WESTERN BLOTTING

SUBMITTED TO : SUBMITTED BY:

MR HARSH NAVRAJPREET

B.TECH
BIOTECH

REG NO-
1040070145

SEC.B
ACKNOWLEDGEMENT
I HAVE SUCCESSFULLY COMPLETED MY TERM
PAPER.FIRSTLY I WOULD LIKE TO THANK MY
SUBJECT TEACHER MR HARSH FOR HIS
GUIDANCE.I WOULD ALSO ILIKE TO THANKS MY
FRIENDS WHO HAS HELPED ME TO FIND
MATERIAL FROM BOOKS AND INTERNET AND
ALSO TO MY CLASSMATES FOR THEIR
COOPERATION.LAST BUT NOT LEAST TO MY
PARENTS FOR THEIR MORAL AND FINANCIAL
SUPPORT.
CONTENT

1 INTRODUCTION

2 SUMMARY

3 HOW DOES WESTERN BLOTTING WORK

4 HOW THE SAMPLE IS PREPARED

5 ANTIBODIES USED
Western Blot

: Western Blot Definition: Western blotting is a technique used to identify and locate
proteins based on their ability to bind to specific antibodies.

Western blot analysis can detect your protein of interest from a mixture of a great
number of proteins. Western blotting can give you information about the size of your
protein (with comparison to a size marker or ladder in kDa), and also give you
information on protein expression (with comparison to a control such as untreated
sample or another cell type or tissue).

Summary: Western Blot Gives You Information on the:

 Size of your Protein

 Expression Amount of your Protein

Western blot analysis can analyze any protein sample whether from cells or tissues, but
also can analyze recombinant proteins synthesized in vitro. Western blot is dependent on
the quality of antibody you use to probe for your protein of interest, and how specific it
is for this protein. Antibodies are now easily obtainable from commercial sources, and
you can purchase one for your protein of interest. If your protein is a novel protein, you
must produce an antibody yourself or get a company to do it for you. In this case you
will need at least a small amount of your protein either purified from cell extracts or
made as a recombinant (ie in vitro or in a recombinant protein expression system).
Antibodies specific to your protein are vital to western blotting as they are able to bind
specifically to your protein of interest instead of the thousands of proteins on your
western blot!

Figure 1. Details of the steps involved in obtaining protein for western blot.
Figure 2. Figure detailing the steps in conducting a western blot.
How Does Western Blotting Work?
. First things first. Obtain a protein sample you want to analyze, such as cell samples.
Lyse the cells to release protein contents. Run these on a gel which separates proteins on
the basis of size. Then transfer these gel proteins onto a membrane using electricity. This
membrane can then be used to probe for proteins of interest using a primary antibody.

What You Need to Western Blot:

 A Protein Sample
 A Good Antibody to Detect your Protein of Interest

Western blot relies on the primary antibody to detect this protein from the thousands of
proteins on your membrane and previously on your gel! (a cell can contain 30,000
different proteins - and these same proteins can even be altered giving you over 300,000
different proteins!). Using an antibody recognizes your primary antibody (a secondary
antibody) you build up a protein-antibody-antibody sandwich! The secondary antibody
has a horse radish peroxidase enzyme which converts a luminol substrate to a light
releasing substance! This light is detected as a spot on film. From this spot you can
determine how much protein is there relative to other spots, or the size of the protein
relative to a size marker that is run also on the gel.

shows a western blot example gel. Lane 1 is a protein size marker ladder which shows
different known sizes of proteins, this can be purchased commercially and the sizes of all
the spots are given in a pamphlet. Lane 3 is a cancer sample and lane 5 is a normal
sample. As you can see the protein in lane 3 has a higher expression than the cancer
sample in lane 5, which is interesting. Also, the protein spots in lanes 3 and 5 are the
same size as the 2nd spot in the size ladder from lane 1. We can then look at the known
protein size from our brochure which we received with the ladder. We then determine
that the size of the protein is 80 kDa. Our protein of interest is also 80 kDa. So we know
that the western blot worked and that the protein is highly expressed in a cancer sample!

To Detect your Protein:

 Buy an Antibody Against Your Primary Antibody Source

 Use an ECL - Chemiluminescence Kit and Film to Get the Results

[
To Prepare Samples for Western Blot:
Whether you have Non-adherent Cells (such as T-cell lines or peripheral blood cells) or
Adherent cells (such as Fibroblast cells or COS cells), you need to remove extraneous
proteins from the media. For non-adherent cells you can gently pellet them with
centrifugation and then wash the pellet with PBS. For adherent cells, simple wash the
flask or dish with PBS prior to western blot.

Western blot analysis examines proteins, and so we want the proteins to be release from
the cells and also prevent the proteins from being cut up by proteases. We need these to
western blot:

- to keep things cold! 4C on ice during cell lysis for western blotting

- use Detergent to break up membranes of cells to release proteins

- Buffer

- Inhibitors (both protease inhibitors and phosphatase inhibitors if we will do western

blot for phospho-proteins)

Detergent - Lysing Cells For Western Blot

SDS and RIPA are detergents that are used to solubilize proteins for later analysis using
western blotting. This is required as many proteins are either inside the cell or located
inside cell membranes, so we need to release these proteins to be able to immunoblot for
them.

Which Antibody should be used for Western Blotting?

You should select an Antibody to use for Western Blot. Usually either mouse
monoclonal antibodies or rabbit polyclonal antibodies are used.

You can use either an antibody without any added groups, or use an antibody which is
conjugated to biotin. These antibodies do not require

Polyclonal vs Monoclonal Antibodies for Western Blot

Monoclonal Antibodies are usually better for Western Blotting due to:

- better signal to noise ratio ( lower background in western blot )

- their higher specificity ( you get less contaminating proteins or Ig )

- overall cleaner results on the western blotting film (less non-specific bands detected)

Polyclonal Antibodies are better suited for Immunoprecipitation:

- they recognize more epitopes

- have higher avidity

[Top]

Lysing Cells for Western Blot:

TIPS before you lyse for western blotting:

If you are going to western blot for protein mass (ie a protein that is not phosphorylated):

- you can lyse in larger volumes (keeping the rest to blot for other proteins)

If you are going to western blot a phospho-protein (or phosphorylated protein) it is

important to do the following:

- make sure you use phosphatase inhibitors ( such as vanadate because phosphatases will
remove the phosphates from your proteins! )

- also lyse cells in the smallest volume possible ( ie lyse in 100 ul loading buffer, this is
done because you can load most of the cells you lysed. This is important as signal is
quite weak when western blotting for phospho-proteins.)

If you are looking at protein-protein interactions:

- do not use SDS as it dissociates protein-protein interactions and thus proteins are
denatured (especially after boiling)

- for western blotting protein-protein interactions, ie native interactions you must use a
less-stringent detergent such RIPA.

Lysing:

- can be done directly on the plate or dish

- pellet the cells

- Keep on Ice and cold - use an ice bucket

- Rule of thumb for how much lysis buffer to use is: 10^5 cells / uL of lysis buffer

- collect in eppendorf tubes and label ( keep these on ice )

Measure total protein before Western Blot Analysis:

- this allows more quantitative analysis if you want to compare treatment conditions in
western blot analysis

- commercial kits are available such as a Bradford assay for measuring total protein
(from Bio-Rad)

- 0.1% of SDS or greater can interfere with protein measurement

Denature Protein Samples :

- add protein loading sample buffer to an aliquot of cell lysate - contains dye (too see the
migration on a gel, 2% SDS)

- add disulfide reducing agent such as beta - mercaptoethanol

- Boil in water bath or heating block (lower temperatures such as mid - 90 degrees
decrease protein aggregation ).

- Poke a hole in cap of each tube to prevent popping

SDS-PAGE Gel Information for Western Blotting
SDS-PAGE Gels are gel matrices which are used to separate proteins by size in the
presence of electric current. Low percentage gels separate larger proteins whereas higher
percentage gels separate smaller proteins better. The problem is that all proteins have a
charged associated with them, and in an electrical current this could cause problems.
This is solved by the addition of SDS to the protein samples. SDS binds to proteins every
few amino acids and neutralizes the charge differences that proteins have. This allows
proteins to be separated by size and not by charge.

SDS-PAGE Gel Preparation for Western Blot

- depending on how much protein you will want to load for western blotting, you should
use small combs or larger combs.

- the percentage of acrylamide is important. Usually 10% acrylamide is used.

- A resolving gel is used at the bottom with a pH of 8.8

- A stacking gel (4-5%) pH 6.8 is used to pack proteins in together after loading

- Polymerization of gels is increased by the catalysts APS and TEMED which speed up
the polymerization reaction (formation and solidification) of the poly-acrylamide gel.

- Load every Sample carefully and slowly to prevent sample leaking out of the lane.

Electroblotting has been a feature of a large number of laboratories for a

(relatively) long time now, and there are a large number of different
apparati around that will efficiently transfer proteins (or other
macromolecules) transversely from gel to membrane. Most of these,
however, are based on the design of Towbin et al. (1979): ie., they
have vertical carbon / stainless steel / platinum electrodes in a large
tank.
Some enterprising newcomers have fairly recently turned the whole issue on its side, and
have started "semi-dry" or "horizontal" blotting. With this technique, one uses two
plate electrodes (stainless steel or graphite / carbon) for uniform electrical field over a
short distance, and sandwiches between these up to six gel / membrane / filter paper
assemblies, all well soaked in transfer buffer. The assembly is clamped or otherwise
secured ON ITS SIDE, and electrophoretic transfer effected in this position, using as
transfer buffer only the liquid contained in the gel and filter papers or other pads in the
assembly.

There are a number of advantages to this procedure over the conventional upright
protocol, not the least of which is that as little as a couple of hundred millilitres of buffer
are all that is needed for electroblotting everal gels, compared to as much as five litres for
some commercial kits. In addition, several gels can be blotted simultaneously; electrodes
can be cheap carbon blocks; less power is required for transfer (and therefore a simpler
powerpack).

Immunoassay

The reason for transferring proteins to membranes from gels is so as to be able to get at
them more efficiently with various probes, as polyacrylamide is not particularly amenable
to the diffusion of large molecules. The most popular type of probe of immobilised
proteins is an antibody of one type or another: the attachment of specific antibodies to
specific immobilised antigens can be readily visualised by indirect enzyme
immunoassay techniques, usually using a chromogenic substrate which produces an
insoluble product. Lately chemiluminescent substrates have begun to be used because of
their greater detection sensitivity. Other possibilities for probing include the use of
fluorescent or radioisotope labels (fluorescein, 125I). Probes for the detection of
antibody binding can be conjugated anti-immunoglobulins (eg: goat-anti-rabbit /
human); conjugated staphylococcal Protein A (which binds IgG of various species of
animal); or probes to biotinylated / digoxigeninylated primary antibodies (eg:
conjugated avidin / streptavidin / antibody).

The immunoassay is normally done by blocking the transfer membrane with a

concentrated protein solution (eg: 10% foetal calf serum, 5% non-fat milk powder) to
prevent further non-specific binding of proteins; this is followed by incubation of the
membrane in a diluted antiserum / antibody solution, washing of the membrane,
incubation in diluted conjugated probe antibody or other detecting reagent, further
washing, and the colorimetric / autoradiographic / chemiluminescent detection.

The power of the technique lies in the simultaneous detection of a specific protein by
means of its antigenicity, and its molecular mass: proteins are first separated by mass
in the SDS-PAGE, then specifically detected in the immunoassay step.

It is also possible to use a similar technique to elute specific antibodies from specific
proteins resolved out of a complex mixture, many of whose components react with a
given antiserum: one can electrophorese a mixture of proteins, cut out a specific band
from a gel or membrane, and use this to fish out specific antibodies from a serum.

Staining of proteins in gels

Staining of proteins in gels may be done using the standard Coomassie brilliant blue (or
PAGE blue), Amido Black, and more lately, silver stain reagents of different kinds. All of
these protocols are relatively long: silver staining requires a number of finicky treatment
and washing steps and takes at least a couple of hours, while other stains are two-step
stain/destain procedures, but require hours (sometimes days) for satisfactory
destaining. Silver staining is (or can be) extremely sensitive - to +1 ng/band) - while of
the other commonly used stains, Coomassie brilliant blue G-250 is probably the best,
but detects only to about 0.3 ug/band. None of these stains are easily reversible, and
most often the protein cannot be recovered intact for other procedures.

However, it is possible to reversibly stain gels prior to blotting by a couple of methods:

the simplest is by simply soaking them in ice-cold 1M potassium chloride: SDS
precipitates as KDS, and proteins are visible as whiter zones in an opaque-to- translucent
white background. The method is not sensitive, however.

Now Lee et al. (C. Lee, A. Levin and D. Branton: Copper staining: a five- minute
protein stain for sodium dodecyl sulfate- polyacrylamide gels; Anal. Biochem. 166,
303-312; 1987) have described an alternative to common protein stains - and to KCl
treatment - which is sensitive, stable, and totally reversible for subsequent recovery of
the protein.

NOTE: you should cut nappy liners / papers to the size of the gel(s) to be blotted, so as to
minimise surface area exposed to the electrode: this reduces the amount of current that
needs to be passed in order to effect transfer (and incidentally reduces heating effects).

CATHODE (-ve electrode) or another layer of nitrocellulose / gel / blotting paper.

Care should be taken to exclude bubbles between gel and nitrocellulose, and between
nitrocellulose and paper.

The assembly is placed in a plastic tray resting on the anode. The ANODE (electrode on
the nitrocellulose paper side of the assembly) is connected to the anode or RED
connector - and the gel side CATHODE to the cathode or BLACK connector - of an
appropriate powerpack. A current of 500mA is passed for 20-30 min to effect transfer.
Transfer is found to be essentially quantitative - for thin assemblies - after
electrophoresis under these conditions. One hour should be sufficient for transfer of all
but the most recalcitrant proteins. If in doubt, blot gels in duplicate, remove one and stain
for protein after 1 hr and continue blotting other.

Washing: blots are washed by shaking in +100 ml/wash, 3x5 min., at room temperature.
20 ml water as the two acids bring with embibed water. The stain works great and
reverses with a normal TS (Tris/saline) wash.”

NB: Recognition of host plant (or other) proteins can be avoided by diluting the
antiserum in buffer containing (for example) a 1:3 mix of sap of healthy plants (plants
crushed 1:1 (w/v) in 0.1 M phosphate, pH 7.0) or other antigen with the buffer used to
dilute the antiserum; and pre-incubating at 37oC for 1 hour. This procedure gets rid of
host plant and E. coli contaminant reactions. Another procedure more generally used in
this laboratory is the removal of anti-host antibodies by plant or bacterial extracts
immobilised on nitrocellulose (or other membrane): simply soak membrane in a
concentrated extract of whatever it is that you want to remove antibodies with +30 min);
block as for normal blot; add diluted antibody suspension to this membrane for +30 min
before use on the Western blot proper, then pour off for use as probe antibody. This
procedure should remove all antibodies responsible for reacting with antigens that
contaminated the inoculum used to immunise rabbits (or whatever).
The Western Blot is Dead and Someone
Forgot to Bury It

A classic laboratory technique, western blotting, has been used by researchers for
decades, and it is nearly obsolete.

He went on to explain that mass spectrometers, instruments that can smash molecules and
then identify them by the size of their fragments, are ready to replace the tedious
procedure.

Western blotting has a lot of flaws. It makes use of antibodies, Y-shaped molecules that
can recognize and latch onto a target protein. Making those sticky molecules can be
tricky, and sometimes they can be fooled -- they might grab onto the wrong protein.

If a team of scientists wants to check some cells for a bunch of different proteins, they
will need several different antibodies -- one for each target. Even if they can buy the
antibodies, their western blot will not tell them exactly how much of each protein was in
their sample.

"Using antibody detection, biologists can find only what they look for," said Mann,
suggesting that they may overlook fascinating data by relying on the old method.

By comparison, mass spectrometers can make extremely precise measurements, and they
can give biologists a look at the big picture -- the levels of nearly every protein in a cell.

Of course mass spec is superior to western blotting, especially with recent technological
developments.
Let's just ignore the fact that a mass spec machine is an expensive and extremely
sensitive piece of equipment that requires high maintenance, properly trained technicians
and adequate IT support. If you want to perform quantitative experiments with mass spec,
you must label your samples or grow your cells on media with expensive isotope-labelled
aminoacids. It's great for people like Matthias Mann who have plenty of MS machines
and expertise to claim that WB is passé but for everyone else who is not as well-funded,
WB is still a viable, useful technique that is less demanding on lab budgets and still
adequate for experimental requirements.

This Matthias Mann has no idea what he is talking about. Mass spec analysis is expensive
as hell (in comparison to Western blotting) and it's sensitivity is also a curse -- every
reagent you use in preparing a sample for mass spec has to be very clean and highly
purified and one has to be extremely careful in how one handles the sample or you end up
just spending a lot of money to find out the sample is full of your skin and hair (keratin is
usually a big hit). Also, a lot of times you don't need to know exactly how much of a
particular protein is in a sample, just relative amounts between experimental samples
which a Western blot can tell you (and you can determine how much protein there is with
other, much less expensive methods).

While a Western blot can be performed by one person in one day in their own lab, you
either need a mass spec facility that is easily accessible or be able to send your sample
out to a facility. In either case, how long is it going to take to get the data from that
analysis? What if the people who run the facility are incompetent?

Mass spectrometry has its place and western blotting has its place.
REFERENCE

1.. www.ars.usda.gov/research/projects/projects.htm
jac.oxfordjournals.org/cgi/content/full/
2.www.thehorse.com/ViewArticle.aspxwww.lapublichealth.org/ACD/AntiBio.htm
3.www.ars.usda.gov/research/projects/projects
4.www.antibioresistance.be/Links.html
5.www.tribuneindia.com/2001/20011104/spectrum/fitness.htm

6.www.lapublichealth.org/ACD/AntiBio.htm
7.www.ars.usda.gov/research/projects/projects
8.www.antibioresistance.be/Links.html