Western Blotting

Western blot technique

a technique for analyzing proteins by separating them by electrophoresis, transferring them in place to a filter or membrane, and probing with specific antibodies.

The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern.

Detection of RNA is termed northern blotting and the detection of post-translational modification of protein is termed Eastern blotting.

Advantages

Specific interaction of antibody and antigen can be directly visualized.

Disadvantage
Technically demanding Expensive

Subject to interpretation
Presence or absence of bands

Intensity of those bands

Western blot analysis of proteins separated by SDS-PAGE gradient gel electrophoresis.

electrophoresis to separate native or denatured proteins by the length of the polypeptide or by the 3-D structure of the protein . The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein.. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

Workshop Timeline
Introduction Prepare the blotting system Block nitrocellulose membrane Incubation with antibody solutions Color development of the blot

Steps in a western blot

Tissue preparation
Gel electrophoresis Transfer Blocking Detection Two step One step Analysis Colorimetric detection Chemiluminescent detection

Radioactive detection
Fluorescent detection Secondary probing 2 2-D gel electrophoresis 3 Medical diagnostic applications

Tissue preparation
1. Lysis buffers
2. Protease and phosphatase inhibitors 3. Preparation of lysate from cell culture 4. Preparation of lysate from tissues 5. Determination of protein concentration 6. Preparation of samples for loading into gels

Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins.
Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing. A combination of biochemical and mechanical techniques including various types of filtration and centrifugation can be used to separate different cell compartments and organelles.

Gel electrophoresis
The proteins of the sample are separated using gel electrophoresis.
Separation of proteins by

Isoelectric point (pI),

Molecular weight, electric charge, combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.

Most common type of gel electrophoresis polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel.

Smaller proteins migrate faster through this mesh Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point

Transfer
The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting

The proteins move from within the gel onto the membrane

Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Both varieties of membrane are chosen for their nonspecific protein binding properties (i.e. binds all proteins equally well). Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie or Ponceau S dyes.

From gel to blot

Polyacrylamide Gel Electrophoresis:
Break protein complexes into individual proteins Separates protein samples based on size

Western Blot Analysis:

Transfer the proteins to a nitrocellulose membrane More stable and permanent Identifies proteins by immunodetection: using specific antibodies against the protein of interest

Prepare to transfer proteins to a nitrocellulose membrane

Mini Trans-Blot Transfer Cell

Place the cassette with gray side down on clean surface

Preparing the Blotting Sandwich

Place one pre-wetted fiber pad on the gray side of the cassette

Place a sheet of filter paper on the fiber pad

Place gel on filter paper taking care to remove air bubbles Place the pre-wetted nitrocellulose membrane on the gel Place the second fiber pad on top Close the cassette firmly DO NOT move gel/filter sandwich

Lock the cassette

Prepare for Electrophoretic Transfer

Place the closed and locked cassette in the electrode module Add the frozen Bio-Ice cooling unit and place in tank Fill the tank with buffer

A stir bar can be added to help maintain the ion and temperature distribution in the tank even

Transfer Proteins from the gel to the nitrocellulose membrane 30 minutes 100V Blotting buffer 1x Tris glycine with 20% ethanol

Blocking
Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive), with a minute percentage of detergent such as Tween 20.

Blocking Buffer
Remove membrane from the blotting sandwich and immerse in 25ml of blocking solution for 15minutes
5% non-fat milk: Prevents the primary antibody from binding randomly to the membrane Phosphate buffered saline (PBS): Provides the correct environment (pH, Salt) to maintain protein shape 0.025% Tween 20: non-ionic detergent that prevents non-specific binding of antibodies to the membrane

Detection
During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour.

Two step
Primary antibody
Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest . Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and specific detection tools that bind the protein directly.

After blocking,

a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/mL) is incubated with the membrane under gentle agitation.
The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight.

Secondary antibody
After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. This is known as a secondary antibody, and due to its targeting properties, tends to be referred to as "antimouse," "anti-goat," etc.

Antibodies come from animal sources (or animal sourced hybridoma cultures); an anti-mouse secondary will bind to almost any mouse-sourced primary antibody.

The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.

Another method of secondary antibody detection utilizes a near-infrared (NIR) fluorophore-linked antibody.

Light produced from the excitation of a fluorescent dye is static, making fluorescent detection a more precise and accurate measure of the difference in signal produced by labeled antibodies bound to proteins on a western blot.
Proteins can be accurately quantified because the signal generated by the different amounts of proteins on the membranes is measured in a static state, as compared to chemiluminescence, in which light is measured in a dynamic state.

One step
This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known protein tags. The primary probe is incubated with the membrane in a manner similar to that for the primary antibody in a two-step process, and then is ready for direct detection after a series of wash steps.

Add primary antibody

Discard blocking solution Pour 10ml of primary antibody onto the membrane and gently rock for 10 minutes Primary antibody will bind to the target protein. Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer Add 50ml of wash leave for 3 minutes on the rocking platform

Add enzyme linked secondary Ab

Discard wash solution Pour 10ml of the secondary antibody onto the membrane and gently rock for 10 minutes Secondary antibody will bind to the primary antibody Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer Add 50ml of wash leave for 3 minutes on the rocking platform

Add enzyme substrate

Discard wash solution Add 10ml of the enzyme substrate (HRP color detection reagent) onto the membrane Incubate for 10 minutes The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody

Rinse and store

Rinse the developed membrane twice with distilled water and blot dry
Air dry for 30min-1 hr and store in lab notebook

Analysis
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis.

Colorimetric detection
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry or spectrophotometry.

Chemiluminescent detection
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody.

Radioactive detection
Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest . The importance of radioactive detections methods is declining, because it is very expensive, health and safety risks are high .

Fluorescent detection
The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.

Secondary probing
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes.. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use.

Medical diagnostic applications

The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). Some forms of Lyme disease testing employ Western blotting. Western blot can also be used as a confirmatory test for Hepatitis B infection.

ELISA vs Western Blotting

ELISA - Quick results - Primary screening - Identifies proteins by antibody specificity only Western Blot Confirm ELISA results - More specific Identifies proteins by both antibody specificity and size

What is so special about SDS?

SDS is a negatively charged detergent.
Disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding protein.

Masks charge on protein so that all proteins act the same as regards charge.
Prevents protein aggregation. Prevents protein shape from influencing gel run.

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