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.