Question

For a Western blot method, please explain each step (simple as possible) in the order below

Steps:

- Sample preparation

- Gel Electrophoresis

-Transfer

-Antibody Probing

-Detecting

-Imaging

-Analysis

Answer 1

How gel is prepared?

PAGE: polyacrylamide gel electrophoresis  is an analytical method used to separate components of protein mixture based on their sites technique is based upon the principle that the charge to molecule will migrate in an electric field to words an electrode with opposite sign the general electrophoresis  technics cannot be used to determine the molecule are weight of biological molecules because the mobility of the substance in the jail depends on to charge and sizes to overcome this biological sample need to be treated so that they require uniform charge then the electrophoretic  depends primarily on size for different protein molecules with different shapes and sizes needs to be denatured so that protein loses their structure

tissue preparation:

Sample may be taken from whole tissue or from cell culture it should be noted that bacteria virus Samples can be the source of protein and the western blotting is not restricted to cellular studies only distorted detergents salts and buffers maybe employed to encourage lysis of the cell and to solubilisation of protein Tissue preparation is often done in cold temperature avoiding protein denaturation

Gel electrophoresis

The protein of the sample or separated using cell electrophoresis separation of protein maybe isoelectric point, molecular weight, electric charge or combination of these factors the principle involved in different in the electrophoretic mobility is of different protein

transvering:

in order to make the protein accessible to antibody detection they are moved from the gel to a membrane made up of nitrocellulose or PVDF the membrane is placed on the top of the gel and stack entire stack is place in the buffer solution which most of the people by Capiilary action bringing the protein with that another method of transvering protein is called electroblotting and uses an electric current to pull protein from the gel to PVDF or nitrocellulose membrane

Blocking

The membrane has the ability to bind to proteins in this case both the target and the antibody protein so there could be some unwanted onted binding blocking of non-specific binding is achieved by placing the membrane in dilutes solution of protein

Does when the antibody is added there is no room on the membrane for it to attach to other than the binding site of specific target protein

detection

it is a two-step method

Primary antibody is applied for protein of interest and is incubated with the membrane the antibody is diluted in the buffer solution along with detergent the 1° antibody specific for the protein interest and at appropriate concentration should not bind any of the other protein on the membrane

Secondary antibody after the rising the membrane to remove unbound 1° antibody secondary antibody after the rising the membrane to remove unbound 1° antibodyA secondary antibody is incubated with a membrane it binds to primary antibody the secondary antibody is typically linked to Biotin or a reporter in signs such as alkaline phosphate Or horse radish per oxide or horse radish per oxide That allows visual identification by process of fluorescence

analysis

The unbound secondary bodies are washed away and the enzyme substrate is incubated with incubator so that the position of membrane bound two degree antibody Will emit light the western blotting is ready for detection of pobe that are labelled to bound to protein of interest

size approximation are taken by comparing the obtained  bands to the marker loaded during electrophoresis