Western Blot

Western Blot is a commonly used experimental method in molecular biology, biochemistry and immunogenetics. Its basic principle is to dye the cells or biological tissue samples processed by gel electrophoresis through specific antibodies. Information about the expression of specific proteins in the analyzed cells or tissues can be obtained by analyzing the location and depth of staining.

Western Blot uses polyacrylamide gel electrophoresis. The detected object is a protein, the "probe" is an antibody, and the "color development" is a labeled secondary antibody.

• Extraction of protein
1. Extract protein from the cell by mechanical or chemical lysis of the cell.
2. Add protease inhibitors to prevent denaturing.
3. Dilute the protein sample in the loading buffer containing glycerol which helps to sink the sample in well.
4. Add tracking dye (bromothymol blue) to the sample to monitor the movement of proteins.
• Gel Electrophoresis
1. Load the sample in the well of SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
2. Separate the proteins on the basis of electric charge, isoelectric point, molecular weight, or combination of these all.
• Blotting
1. Place the nitrocellulose membrane on the gel. Transfer the separated protein from gel to nitrocellulose paper by capillary action.
2. Use electro-blotting for fast and more efficient transfer of desired protein from the gel to nitrocellulose paper.
• Blocking
  Use casein or Bovine serum albumin (BSA) to make the membrane non-specifically saturated or masked before adding the primary antibody.
• Antibody incubation
1. Form Ag-Ab complex with the primary antibody and desired protein.
2. Bind with Ag-Ab complex using the secondary antibody.
• Protein detection and visualization
1. Incubate the reaction mixture with the specific substrate to visualize the enzyme action.
2. Convert the substrate to visible colored product with enzyme for visualizing band of color in the membrane.

Fig. 1 The basic flow chart for western blot technology.

• For qualitative detection of single proteins and protein modifications such as post-translational modifications.
• For medical diagnostics such as HIV testing, BSE testing, tularemia diagnosis, testing for some forms of Lyme disease, confirmatory testing for hepatitis B infection and HSV-2 (herpes type 2) infection, FIV+ status in cats.
• Applied to the World Anti-Doping Agency.

Laboratory Centrifuges

A centrifuge is any device that applies a sustained centrifugal force—that is, a force due to rotation. The widest use of centrifuges is for the concentration and purification of materials in suspension or dissolved in fluids. Suspended particles denser than the suspending liquid tend to migrate toward the periphery, while those less dense move toward the centre.

Homogenizers / Sonicators

Homogenizers, commonly referred to as "sonicators", disrupt tissues and cells through cavitation and ultrasonic waves. Basically, a homogenizer has a tip which very rapidly vibrates, causing bubbles in the surrounding solution to rapidly form and collapse. This creates shear and shock waves which tear apart cells and particles. Homogenizers / Sonicators are great for breaking apart cells and subcellular structures in suspension. They are not good for homogenizing intact tissue.

Electrophoresis Systems

Electroporation refers to the use of short high voltage pulses to overcome cell membrane barriers. Transient and reversible breakdown of the membrane can be induced by applying an external electric field, which just surpasses the capacitance of the cell membrane. Electroporation is now used to deliver a large variety of molecules: from ions to drugs, dyes, tracers, antibodies, oligonucleotides to RNA and DNA.

Ice Maker and Dry Ice Maker

Ice makers are used to produce ice from water or “dry ice” from liquid nitrogen. Most laboratories have access to an ice machine, since ice is used to keep samples and reagents cold during experiments or other lab work. Ice makers are typically large floor-model units that are plumbed directly to a water source. Ice makers vary in size based on the amount of ice they can produce and store.

Laboratory Rockers and Shakers

A rocker or shaker is a piece of laboratory equipment used to mix, blend, or agitate substances in a tube or flask by shaking them. These important tools are used for a variety of applications including cell culture, DNA extraction, mixing reagents, and staining gels and blots. A shaker contains an oscillating board that is used to place the flasks, beakers, or test tubes.

Nitrocellulose Membrane

Nitrocellulose membrane refers to a sticky membrane used for immobilizing nucleic acids and proteins. It is a common matrix used in Western blotting due to the high protein-binding affinity. Nitrocellulose membranes are available in 100% pure forms of nitrocellulose with a high surface area, which is uniform. One of the main advantages of nitrocellulose membranes is its very low background as it can be easily blocked.

Gel Imaging Systems

Gel imaging or gel documentation (gel doc) systems are used for the analysis of proteins, antibodies and nucleic acid immobilized in polyacrylamide or agarose gels, membranes or microarrays. Systems come in a variety of configurations depending on throughput and sample type. It is widely used in: monoclonal and polyclonal antibody binding affinities, gel and blot imaging, colony counting, immunoassay, multiplex protein detection and other fields.

Spectrophotometers

Spectrophotometer is an optical instrument that measures the intensity of light in relation to the wavelength. It can measure just about anything such as: plastics, paper, liquid, metal, fabric and so on. A spectrophotometer is used to check for the quality of product in a production facility and identify and quantify microscopic samples in analytical laboratories.

Chemiluminescence Imaging Systems

An automatic chemiluminescence imaging system is a fully automatic computer-controlled professional shooting and analysis system for real-time high-definition dynamic imaging. It is used for chemiluminescence detection of Western Lightning, ECL\ECL plus, CDP-Star, SuperSignal, CSPD, LumiGLO, and other luminescent substrates.

STEMart provides you with a variety of western blot equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for western blot, please feel free to contact us.