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The Western blot technique, also known as immunoblotting, is a highly-sensitivity and semi-quantitative molecular technique used for the detection and analysis of a specific protein or protein profile. Proteins are separated according to their net charge and molecular weight and are then transferred to a solid membrane, in which a primary and secondary antibody bind to the target protein and are then visualized using different detection systems.
Western Blotting, also known as immunoblotting when first described in 1979, is a ubiquitous method in the modern life science laboratory. From cell biology to protein purification and characterization, western blotting remains an essential protein analytical technique that is fundamental to protein research.
A Western blot is simply a way to identify proteins on a polyacrylamide gel. Proteins, separated by size on a polyacrylamide gel, are transferred to a membrane such that their pattern in the gel is retained, like making a photocopy. That “blot” is then probed using “primary” antibodies to a specific protein or proteins of interest, and developed by addition of secondary antibodies, which recognize the primaries. These secondary antibodies are labeled with either a radioisotope, fluorophore, or enzyme, all of which allow the protein's positions to be identified.
For Western blots in particular, the optimization of various steps and a great deal of experience working with the technique are key to gathering quality data that is reliable and reproducible. As a multistep process, the technique inherently houses many places in which variability can be introduced during data collection. Some of the critical factors for achieving accurate immunoblotting data include sample preparation, loading controls, and choice of reagents and buffers.
There are two main types of Western Blot, Northern and Southern Western Blot.
This technique detects specific RNA molecules using a DNA or RNA probe of a similar sequence. This is followed by electrophoresis and detection on the membrane with a radioactively or chemically labelled detection reagents.
This technique detects specific DNA molecules using a DNA or RNA probe of a similar sequence. This is followed by electrophoresis and detection on the membrane with a radioactively or chemically labelled detection reagents.
1. Both takes place under in-vitro conditions
2. Require antigen-antibody interaction
3. They have reproducible results
4. They have improved data quality
5. Both can be used in detect the presence of certain proteins and antibodies.
|Detects the presence of antigen and antibody in cell lysates, serum, etc.||Detects the presence of a specific protein and its MW from a mixture of proteins|
|Relatively less time-consuming||Time-consuming, delicate process, costly|
|Qualitative and quantitative||Qualitative and sometimes semi-quantitative|
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Below are some Western Blot terms and their descriptions
Sample preparation: The first step in a Western blotting procedure is the preparation of a cell or a tissue lysate, using a lysis or extraction buffer. This lysate contains the cellular contents released from the broken cell membranes. Protein(s) of interest are then solubilized and separated with gel electrophoresis. Find out more about sample preparation.
Mechanical methods of cell or tissue lysis: Proper lysis and release of cellular contents from animal and herbal tissues, cell cultures and bacteria, etc. is essential in obtaining high protein yield and good quality results. Common mechanical methods used are sonication, homogenization using abrasives, and disintegration with glass or metallic beads.
Chemical methods of cell or tissue lysis: Proper lysis and release of cellular contents from animal and herbal tissues, cell cultures and bacteria, etc. is essential in obtaining high protein yield and good quality results. The chemical methods include the use of buffers capable of solubilizing the proteins, since they contain ionic detergents such as sodium dodecyl sulfate (SDS), deoxycholate and cetyl trimethylammonium bromide (CTAB).
Sonication: Sonication is the third class of physical disruption commonly used to break open cells. The method uses pulsed, high frequency sound waves to agitate and lyse cells, bacteria, spores and finely diced tissue.
Mortar and pestle: Manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of the tensile strength of the cellulose and other polysaccharides comprising the cell wall, this method is the fastest and most efficient way to access plant proteins and DNA.
Freeze-thaw method: The freeze-thaw method is commonly used to lyse bacterial and mammalian cells and involves freezing of cell suspension and then thawing it at room temperature or 37°C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing.
Lysis buffers: The cell membranes must be broken open to release the cellular contents using, lysis or extraction buffer, and the protein of interest needs to be solubilized before it can be separated with gel electrophoresis. Reagents utilized to efficiently solubilize cellular proteins in order to run them for electrophoresis are called lysis buffers, and can be ionic or non-ionic in nature. Ionic buffers such as, Sodium Dodecyl Sulfate and CTAB are considered the harshest and are likely to give higher yield. The composition of lysis buffer substantially affects the results of quantitative immunoblotting.
Protein quantification: Before performing the electrophoresis, the protein content needs to be quantified in order to homogenize the amount deposited in the well of the gel and to determine the protein concentration. A large amount of protein can saturate the immunodetection and yield unspecific results.
Lowry Protein Assay: This method is characterized by the use of Folin's reagent and copper. It is detected in a wavelength of 750 nm, and the color intensity is based on the protein concentration.
Bradford Protein Assay: This method uses Coomassie Brilliant Blue dye, which reacts in the presence of proteins, and the color change is detected in the wavelength range of 465-595 nm.
Bicinchoninic Acid Assay: The protein assay with bicinchoninic acid (BCA) is highly sensitive and combines the reaction of proteins with Cu2+ ions in an alkaline method resulting in the formation of purple color. Try our BCA assay today.
Loading Control: To complement total protein estimates, immunoblots typically include loading control proteins which provide a secondary check that roughly equal amounts of cellular materials have been added. They are used for the qualitative confirmation of overall protein abundance across the gel. Housekeeping genes are often used as loading control, and are essential for the reproducibility of your data.
Phosphate Buffered Saline: Phosphate Buffered Saline (PBS) is routinely used as a wash buffer in Western blot and immunoprecipitation (IP) procedures, as well as during sample preparation of cell lysates. It can also be used to disengage attached and clumped cells while also helping to maintain a constant pH. However, in the case of using Alkaline Phosphatase as a reported in Western Blots, it is recommended that PBS is replaced with Tris Buffered Saline (TBS).
Nitrocellulose Membrane: Nitrocellulose membranes are a popular matrix used in protein blotting because of their high protein-binding affinity, compatibility with a variety of detection methods (chemiluminescence, chromogenic, and fluorescence), and the ability to immobilize proteins, glycoproteins, or nucleic acids. Boster’s Nitrocellulose Membrane, 0.45 µm, 9 cm x 10cm is used for a wide range of protein molecular weights and nucleic acids >500 bp. Order our Nitrocellulose Membrane today.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE): It is the most commonly used technology to obtain high resolution analytical separation of mixtures of proteins. It involves initial denaturation of component proteins with an anionic detergent that also binds to them, imparting to all proteins a negative charge proportional to their molecular mass. This step is followed by electrophoresis through a porous acrylamide gel matrix that separates proteins with excellent resolution on the basis of molecular mass.
PVDF (polyvinylidene difluoride): Polyvinylidene difluoride (PVDF) membrane is ideal for western blotting applications as well as for amino acid analysis and protein sequencing of small amounts of proteins. In addition, PVDF membranes can be used, stripped and re-probed without a loss of sensitivity or increased background. PVDF membranes tend to be used more frequently because their protein binding strengths exceed those of nitrocellulose membranes.
Coomassie Brilliant Blue (CBB): CBB is widely popular staining method of proteins which makes them easy to visualize and is cost-effective. It binds to proteins non-covalently through ionic and hydrophobic interactions.
Ponceau S: Ponceau S solution can be used for rapid staining of protein bands on PVDF and nitrocellulose membrane. Since the dye of this reagent is with negative charge, it can combine with amino-acid residue with positive charge. With advantages of convenient use, low background and high sensitivity, this product can detect protein at minimum 250ng, but it is not suitable for protein detection on nylon membrane.
Silver Stain Kit: Silver Staining Kit uses ammoniacal silver chemistry and glutaraldehyde sensitization to produce a highly sensitive silver stain, capable of detecting much lower levels of protein than standard Coomassie or Colloidal Blue techniques. Clear background makes sample identification unambiguous and provides publication-quality gels.
Blocking: Blocking is a very important step of western blotting, as blocking reagents reduce the nonspecific binding of antibodies onto the membrane and hence, reduce the background. Blocking is often made with 5% BSA or nonfat dried milk diluted in TBST to reduce the high background signal. Nonfat dried milk is often preferred as it is inexpensive and widely available. You have to try several options to see which blocking buffer works best for your system.
Antibody binding: After blocking, the membrane is incubated with a solution containing the primary antibody which recognizes and binds to the epitope of the target protein. This is followed by a washing step, a secondary antibody is added which has the ability to recognize the primary antibody and is conjugated with an enzyme such as Horse Radish Peroxidase (HRP) or Alkaline Phosphatase (AP) which helps in the detection phase.
RIPA Lysis Buffer: RIPA Lysis Buffer reagent is a complete cell lysis reagent popularly used for cultured mammalian cells. RIPA lysis buffer is highly compatible with immunoassays, protein purification procedures, immunoprecipitation, and western blotting. RIPA buffer ensures efficient cell lysis and protein solubilization preventing protein degradation and interference with protein immunoreactivity and biological activity. Try Boster's RIPA buffer for your Western Blot.
Stripping Buffer: WB Stripping Buffer allows removing primary and secondary antibodies from probed Western blot membranes without removing or damaging the immobilized antigen. It permits more time efficient experiments that use less sample by reusing membrane without having to re-run gels and blots.
Mounting Medium: Mounting medium is designed to protect fluorescent dyes from fading during fluorescence microscopy experiments. It usually comes ready-to-use, just apply a drop to the sample and add a coverslip. There is no quenching of fluorescent signal of mounted slides after storing for two weeks, protected from light.
Proper control design is essential to western blot. It will guarantee accurate and specific test result by identifying various problems quickly and precisely. There are 5 common types of controls seen in Western blot experiment design.
How to choose the gel percentage, SDS-PAGE gel percentage calculator, the relationship between gel percentage and pore size
Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel. Typically resolving gels are made in 5%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer and ladders will be placed) is inserted. The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used. Changes on the buffer system of the gel can help to further resolve proteins of very small sizes
Check the table below for common protein sizes and their recommended gel percentages.
|Range of molecular weight (KD)||Concentration of gel (%)|
|10 - 30||12|
|30 - 100||10|
|100 - 500||8|
Representing at least 8-9% of all protein-related publications within the last decade, Western Blotting is estimated to be the most used technique across a broad range of scientific and clinical disciplines. Their ability to confirm the identity/presence of a specific protein both by size and through the binding of an antibody provides additional information not readily gathered from other key immunological laboratory techniques. Their results are less ambiguous, making them suitable as a key supplemental screen to be used in combination or provide confirmation of results from other techniques (ELISA, IHC, etc.)
There are three common detection methods for western blots
Chemiluminescence is a popular detection method that uses hydrogen peroxide to oxidize luminol, producing a transient burst of photons that must be detected using autoradiography or a digital imager.
Chemifluorescence is an alternative labelling and detection method for molecular biology and biochemistry that combines fluorescence and chemiluminescence. Whilst chemiluminescence generates light based on an enzymatic reaction, chemifluorescence attaches a fluorescent molecule to either the secondary or tertiary antibody which requires excitation via a light source.
Fluorescence involves using secondary antibodies conjugated with a fluorescent molecule (fluorophore). When the fluorophore is excited by a light source, this causes the release of photons as the excited molecule returns back to its normal state which is then detected in the form of light. The light emitted from fluorophores is consistent and directly proportional to the amount of protein on the membrane. Researchers performing fluorescence-based Westerns have the ability to combine two or more secondary antibodies to detect multiple proteins at once, a process called multiplexing.
Coomassie Brilliant Blue (CBB): CBB is widely popular staining method of proteins which makes them easy to visualize and is cost-effective. It binds to proteins non-covalently through ionic and hydrophobic interactions. Coomassie blue has a similar sensitivity to amido black. Coomassie blue-stained proteins (>50 ng/band) appear as dark blue bands against a light blue background.
Ponceau S: Ponceau S solution can be used for rapid staining of protein bands on PVDF and nitrocellulose membrane. Since the dye of this reagent is with negative charge, it can combine with amino-acid residue with positive charge. With advantages of convenient use, low background and high sensitivity, this product can detect protein at minimum 250ng, but it is not suitable for protein detection on nylon membrane. Transferred proteins (>250 ng/band) appear as red bands on a pink background. Major advantages of Ponceau S staining are that it is simple, rapid, and easily reversible.
Amido black: Amido black is used to stain proteins on blot transfer membranes. Transferred proteins (>50 ng/band) appear as dark blue bands on a light blue background. Amido black has a sensitivity similar to that of Coomassie blue, but it stains faster. It is the preferred stain for protein sequencing and in situ cleavage of proteins for determining internal sequences because the mild staining and destaining conditions minimize the likelihood that any protein will be extracted during the procedures. Amido Black aids protein visualization even at low concentrations. It has applications in forensics due to its ability to stain blood proteins in fingerprints.
Colloidal gold: Colloidal gold is a highly sensitive stain and commercial, ready to use stains are available. Transferred proteins (>2 ng/band) will appear as red bands on a pink background. A higher signal may be obtained with alkali treatment of the membrane prior to staining by washing the membrane with 1% KOH followed by several rinses with phosphate-buffered saline (PBS).
Colloidal silver: Colloidal silver is a more economical stain than colloidal gold. The staining procedure is rapid, although the ferrous sulfate solution must be prepared immediately before use. Transferred proteins (>5 ng/band) appear as black bands on a light brown background for nitrocellulose and on a dark background for PVDF.
India Ink: India ink is used to stain electroblotted proteins on blot transfer membranes. Transferred proteins (>5 ng/band) appear as black bands on a gray background. Sensitivity may be enhanced by brief alkali treatment of the membrane with 1% KOH followed by several rinses with PBS.
Viral Infections and Western Blot: Immunoblotting has been used in clinical diagnosis for serosurveillance and as confirmatory tests for human viral infections. Western blotting assay can detect viral proteins or antiviral antibodies. HIV infection can be identified by the detection of HIV-specific antibodies in serum or plasma. Western blot has been traditionally used as a confirmatory test after a positive ELISA result. In the Western blot test, the sample is separated with an electrical current and transferred onto a piece of blotting paper. An enzyme is added to cause color changes that signal the presence of HIV antibodies. Western blotting assay was also used for the detection of anti Chikungunya virus antibody in human serum.
Detection and characterization of proteins in Alzheimer: Alzheimer’s disease is a neurodegenerative disorder characterized clinically by progressive loss of intellectual function. With the help of Western Blot, scientists have been able to purify and characterize a specific protein present in much higher concentration in certain brain regions of Alzheimer patients than in normal brain.
Western Blot as a confirmatory test for Lyme disease: Lyme disease is a multisystem disorder which may involve dermatological, musculoskeletal, nervous system or cardiac manifestations. The causative agent is a spirochete called Borrelia burgdorferi. Western Blot is a confirmatory test for the detection of Lyme disease after a positive ELISA result and helps in establishing a definitive diagnosis.
Learn the concept behind Western blotting, a technique used to detect specific proteins in a given sample. It usually involves two major processes, SDS-PAGE and protein blotting and testing.Learn Western Blot Principle
Familiarize yourself with the concepts behind our best practices on Western Blotting optimization. Find out how to optimize every aspect of your experiment to yield the best results.Learn Western Blot optimization
Follow our step-by-step Western blotting protocol from reagent preparation to detection using Boster Bio reagents. Check out our protocols to learn how to get the best results.Check out Western Blot Protocol
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