WHAT IS A WESTERN BLOT?

IS THIS A WESTERN BLOT?

NO!

What’s a REAL western blot?

• Analytical technique used to identify and locate specific proteins in a sample (containing mixture of proteins) based on their ability to bind to specific antibodies

• Gives information on:- Size of protein (with comparison to a size marker or

ladder in kDa)- Expression amount of protein(with comparison to a

control such as untreated sample or another cell type or tissue)

Steps

1. Tissue preparation

2. Gel electrophoresis to separate proteins

3. Transfer to a membrane (nitrucellulose or PVDF) where they are detected by antibodies specific to the protein

4. Blocking

5. Detection

Tissue preparation

• Samples can be taken from whole tissues, cell culture, bacteria, viruses, environmental samples etc that are homogenized in a buffer to protect the protein of interest from degradation- Solid tissues broken down mechanically (eg by

blender)

• Detergents, salts or buffers may be added to encourage lysis (breaking of cell membrane) and solubilize proteins

• Done at low temperatures to prevent protein denaturation

Gel electrophoresis

• Proteins of sample separated

• Nothing much to explain

Transfer

• Proteins moved from within the gel to a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF)

• Membrane is placed on top of gel, with a stack of filter papers placed on top of that

• Entire stack is placed in a buffer solution, which moves up the paper through capillary action, bringing the proteins up with it - proteins are exposed on a thin surface layer for

detection• Another method is called electroblotting, where

an electric current pulls the proteins from the gel to the membrane

Blocking

• As the membrane is able to bind protein, steps are taken to prevent interactions between the membrane and the antibody used for detecting the target protein- Membrane placed in in a dilute solution of protein with a minute

percentage of detergent- Protein in the dilute solution attaches to the membrane in all the

places where the target protein has not- No “room” for added antibody to bind onto the membrane other

than the binding sites of the target protein- This ensures clearer results and eliminates false positives

Detection

• Membrane is "probed" for protein of interest with a modified antibody - Antibody linked to a reporter enzyme- Drives a colourimetric reaction and produces a colour

when exposed to an appropriate substrate • This takes place in 2-step process:a.A primary antibody is added at an appropriate

dilution and incubated with the membrane. It will bind to the target protein if it is present.

b.Membrane rinsed to remove unbound primary antibody. In order to detect the antibodies which have bound, a second antibody (or “conjugate”) is added. These are anti-immunoglobulin antibodies.

Detection (cont’d)

• After excess second antibody is washed off, a substrate is added- precipitates upon reaction with the conjugate

resulting in a visible band where the primary antibody bound to the protein

• An isotope-labeled primary antibody can also be used, which can be detected directly by X-ray film and does not require the secondary antibody

Applications

• Medical diagnosesHIV test through human serum sampleBovine spongiform encephalopathy (the

cheem name for mad cow disease)Lyme diseaseHepatitis B

Example of western blot resultHIV Western Blot Test

• The Western blot positive control lane contains proteins from patient sera as well as HIV proteins. HIV positivity can therefore only be confirmed by the presence of certain types of proteins

• No bands present : Negative

• Bands at either p31 OR p24 AND bands present at either gp160 OR gp120 : Positive

• Bands present, but pattern does not meet criteria for positivity: Indeterminate

Lane 1, HIV+ serum Lane 2, HIV- serum Lane A, Patient A Lane B, Patient B Lane C, Patient C

THE END!!!!

Any questions?

References

• http://en.wikipedia.org/wiki/Western_blot

• http://www.molecularstation.com/protein/western-blot/

• http://www.biology.arizona.edu/IMMUNOLOGY/activities/western_blot/west1.html

• http://askabiologist.asu.edu/expstuff/mamajis/western/western.html