Question

Which sequence is the correct order for performing a Western blot?

A- Separate protein using acrylamide gel electrophoresis with SDS

B- Wash off the unbound antibodies

C- Block the nitrocellulose with milk proteins

D- Transfer proteins onto nitrocellulose using a Western Blot transfer apparatus

E- Wash the membrane and incubate with primary antibodies against HAS

F- Incubate the membrane with the conjugate color reactants

Group of answer choices

1. A, B, C, D, E, F

2. A, D, C, E, B, F

3. A, C, D, F, E, B

4. D, A, D, B, E, F

Answer 1

Ans.

• Western blotting is a method of protein detection in a given sample by transfering the biological sample from a gel to the membrane and then detection of protein on membrane surface.
• There are many sequence vise steps in western blotting ;

1). Seperation of protein using SDS-PAGE

• It is the first step of western blotting procedure and involves seperation of macromolecules in a sample.
• Charged components of sample like protein or DNA get seperated depending upon their physical properties, when an electric current is run through the gel.
• It is a commonly used technique for the analysis of a single or multiple proteins in a given sample.

2). Transfer of proteins onto a membrane

• This involves transfering the proteins from the gel onto membrane.
• Many methods are available for this process like diffusion, electroelution...etc.
• Electroelution or electrophoretic transfer is the most common method of transfering the protein because of it's high speed and good efficiency of transfer.

3). Blocking non-specific sites

• It is a commonly used approach in western blotting method that involves blocking of free membrane surface to stop non-specific binding of antibody in the proceeding steps.
• Many types of blocking agents like milk, bovine serum albumin, highly purified proteins...etc can be used depending upon situation and preferences.
• Most of the time researchers prepare fresh blocking buffers in the labs.

4). Washing of membrane and incubating with primary antibody

• It is a necessary step of western blotting.
• Washing of membranes results in the removal of unbound reagents and background reduction, thus improving the ratio of signal-to-noise.
• Most commonly used wahing buffers are TBS and PBS which may be used depending upon the situation.
• The antigen to be detected in the given sample determines the choice of primary antibody.
• A tagged secondary antibody is almost always used as an ultimate means of antigen detection.
• Many types of secondary antibodies are available for use in the detection of western blots.

5). Washing off unbound antibodies.

• This step removes any unnecessary, unbound antibody from the membrane which may otherwise cause a disturbance during detection.
• Presence of unbound antibodies may produce a background effect.

6). Incubating the membrane with conjugate color reactants

• Many different type of tags are available for association with primary or secondary antibody.
• Enzymes and fluorophores are the most commonly used color tag nowadays.

So, the correct order for performing a western blot is - A, D, C, E, B, F