Basic western blotting procedure in reference to HIV test

Basic Blotting Procedure
Basic western blotting procedure includes the following steps;

1. Sample Preparation
2. Gel Electrophoresis
3. Membrane Transfer
4. Blocking Non-specific Binding
5. Addition of the Antibody
6. Detection
7. Troubleshooting

Western blotting
 For basic western Blotting procedure the following steps must have to follow;

1.Proteins separated by gel electrophoresis ,usually SDS-PAGE.(polyacrylamide gel electrophoresis+++) are transferred to a sheet of nitrocellulose membrane.

2.The proteins retain the same pattern of separation they had on the gel.

3.The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose.

4.An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

5.The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

+++Adv of PAGE

i.Inertness to chemicals

ii.Superior resolution

iii.Stability over wide range of ph, temperature, ionic strength.

1.Sample Preparation

•An unlabeled solution of proteins, frequently an extract of cells or tissues, is first prepared in a gel electrophoresis sample buffer.

•In some cases, the sample to be blotted has been derived from an immunoprecipitation.

2.Gel Electrophoresis:

•Protein first resolved by gel electrophoresis.

•acrylamide concentration appropriate for the anticipated molecular weight.

•acrylamide solution is degassed prior to casting gel.

•Fresh ammonium persulfate and TEMED should be used to catalyze gel polymerization.

•Rinse wells thoroughly before applying sample to gel.

•Apply 10–50 µg of total cell or tissue lysates or 0.1–1.0 µg of a purified protein in 1x SDS-PAGE Sample Buffer per well.
                                
•Samples should be heated at 50–65°C for 10–15 minutes prior to loading gel. Samples should not be boiled as proteins containing significant stretches of hydrophobic amino acids (such as membrane proteins) tend to aggregate when boiled.

•Run pre-stained molecular weight markers in one well (to monitor the transfer of protein from the gel) . Help to orient the gel during the transfer procedure.

•run unstained molecular weight standards as well for accurate determination of molecular weight.

•Electrophorese the sample through the polyacrylamide gel.

•Stop electrophoresis when the bromophenol blue dye front reaches the bottom of the geland these are the 1st steps of Basic western blotting procedure in reference to HIV test.

3.Membrane Transfer (Western blotting)

After doing gel Electophoresis in Basic western blotting procedure in reference to HIV test this steps followed as;
•Transfer: by either capillary blotting or by electroblotting (semi-dry and tank transfer systems),

•a sandwich of gel and Nitrocellulose membrane is compressed in a cassette,

•immersed in buffer between two parallel electrodes,

•A current is passed at right angles to the gel, which causes the separated proteins to
 electrophorese out of the gel and onto the solid support membrane,

•Once the proteins have been transferred to the solid support membrane, the membrane is referred to as a “blot”.

•Efficiency: dependent on the protein binding capacity of the membrane used, the transfer method and conditions employed as well as the nature of the antigen itself (size (MW) & hydrophobicity ).

To transfer a protein from a gel to a membrane:

•Following SDS-PAGE, the gel is prepared for electroblotting using a standard tank transfer,

transfer “sandwich” with layers in order from cathode (-) to anode (+)

1) sponge

2) filter paper (3 sheets) soaked in transfer buffer

3) gel

4) membrane

5) filter paper (3 sheets) soaked in transfer buffer

6) sponge

Here are some of the photos of Basic western blotting procedure in reference to HIV test which is shown in another link.

4.Blocking Non-specific Binding:

•Prior to addition of primary antibody, the membrane must be incubated with a suitable blocking protein solution to block remaining hydrophobic binding sites on the membrane.

•This will reduce background and prevent binding of the primary antibody to the membrane itself.

•Typical blocking solutions include 10% (w/v) BSA, or 5% non-fat dried milk in Tris or phosphate buffered saline.

•It is important to be certain that the blocking solution does not contain antigens that may be recognized by the primary or secondary antibody.

•Incubation in blocking solution for 30 minutes at 37°C is sufficient to block membrane.

5.Addition of the Antibody

This step is the important step of Basic western blotting procedure in reference to HIV test and it is done as in the following steps;
•Dilute the primary antibody in Tris or Phosphate buffered saline.

•Unless non-specific reactivity is observed or anticipated, it is not necessary to add blocking protein to the primary antibody.

•Following incubation in primary antibody, the blot is washed in several changes of wash buffer (Tris or Phosphate buffered saline with 0.1% Tween 20) before addition of secondary antibody.

•Follow by incubation with a labeled secondary antibody (as above).

6.Detection

•The method of detection is dependent upon the label that has been conjugated to the primary (or secondary) antibody.

•most common label : alkaline phosphatase or horseradish peroxidase, (chromagen to a colored precipitate at the site of antibody binding)

•Alternatively, chemiluminescent substrates may be employed which emit light (captured on x-ray film) upon conversion by the enzyme.

Detection… Other labels include:

•125I-labeled secondary antibody, which can be detected using a photographic film.

•125I-labeled Protein A. In this case Protein A is used instead of a secondary antibody, as it will bind to the Fc region of IgG molecules.(Basic western blotting procedure in reference to HIV test)

•Gold-labeled second antibody. The minute gold particles are directly visible as a red color when they are bound with the second antibody to the primary antibody.

•The enzyme used is usually alkaline phosphatase or horseradish peroxidase.

western blot results photos

Here completes the chapter named Basic Western Blotting procedure in reference to HIV

Protiens considered to be Antigens in WESTERN blotting.see another blog