Western blotting is method to detect proteins as well as posttranslational modifications on proteins. Gentaur produces rabbit polyclonal antibodies to obtain the molecular weight of your target protein from a complex sample. To find your target protein you need to compare the supposed molecular weight you found through bibliography to a protein weight ladder in kDa or kiloDalton after electrophoresis.
What is the first step for Western blot?
You start with Protein Electrophoresis:
The protein is migrated towards a positive Anode in a sds polyacrylamide gel. All proteins are visualized with InstantBlue.
Gentaur supplies superior 10×10 cm Atto glass precast polyacrylamide gels that will exchange faster heat than plastic precast gels. You will be able to apply higher voltage to your Consort power supply and have faster results without the gel melting from the heat production.
How we detect the sample in Western Blot?
Detection of your target protein involves two antibodies, a primary antibody against your target protein and a secondary antibody against the rabbit. Gentaur produces rabbit polyclonal in a 51 days protocol. The secondary anti rabbit antibody is conjugated to an enzyme or a fluorophore.
Western Blot DETECTION:
More sensitive ECL Light emission occurs only during the enzyme-substrate reaction and, therefore, once the substrate is exhausted, the signal output ceases. In contrast, less specific colorimetric substrates, such as DAB, produce precipitate that remains visible on the membrane even after the reaction has terminated.
How many detection techniques are there?
Western Blot uses 3 ways to detect the protein:
Chemiluminescence Western blots are probed with a primary antibody against the target protein, followed by a secondary antibody labeled with HRP (horseradish peroxidase) enzyme. A chemiluminescence substrate for the HRP enzyme is carefully applied to the blot, and light is emitted when the HRP enzyme modifies the substrate.
Western Blot PROTEIN TRANSFER in a Blotter
The proteins when submitted to a vertical electrical charge will travel through your SDS page Protogel in an electrical field. Your proteins will be transferred in an electrical field from the gel onto PVDF or Nitrocellulose, a membrane that “blots” the proteins from the gel.
Transfer can be done using a wet or semi-dry Blotting System. Wet transfer is less prone to failure due to drying of the membrane, and is especially recommended for large proteins.
In a wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge > paper > gel > membrane > paper > sponge) and all are clamped tightly together to ensure that no air bubbles form between the gel and membrane. A transilluminator is used to read the results of the western blot electrophoresis.
You can reuse your blot membranes by using ADI’s Western blot recycling kit to strip antibodies multiples times form Nitrocellulose or PVDF membranes.