1. WHAT IS A WESTERN BLOT?

2. IS THIS A WESTERN BLOT? 

3. NO!

4. 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)

5. Steps Tissue preparation Gel electrophoresis to separate proteins
Transfer to a membrane (nitrucellulose or PVDF) where they are detected by antibodies specific to the protein
Blocking
Detection

6. 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

7. Gel electrophoresis Proteins of sample separated
Nothing much to explain 

8. 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

10. 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

11. 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 primary antibody is added at an appropriate dilution and incubated with the membrane. It will bind to the target protein if it is present.
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.

12. 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

14. Applications Medical diagnoses HIV test through human serum sample
Bovine spongiform encephalopathy (the cheem name for mad cow disease)
Lyme disease
Hepatitis B

15. Example of western blot result
HIV 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

16. THE END!!!!
Any questions?

17. References http://en.wikipedia.org/wiki/Western_blot