Western Blotting and Molecular Biology: June 2013

ELISA/Western blot tests for HIV

HIV ELISA/Western blot is a set of blood tests used to diagnose chronic infection with human immunodeficiency virus (HIV).

How the Test is Performed

A blood sample is needed. For information on how this is done, see: Venipuncture.

How to Prepare for the Test

No preparation is necessary.

How the Test Will Feel

When the needle is inserted to draw blood, some people feel moderate pain, while others feel only a prick or stinging sensation. Afterward, there may be some throbbing.

Why the Test is Performed

Testing for HIV infection is done for many reasons, including:

Screening people who want to be tested
Screening people in high-risk groups (men who have sex with men, injection drug users and their sexual partners, and commercial sex workers)
Screening people with certain conditions and infections (such as Kaposi's sarcoma or Pneumocystis jirovecii pneumonia)
Screening pregnant women to help prevent them from passing the virus to the baby
When a patient has an unusual infection

Normal Results

A negative test result is normal. However, people with early HIV infection (termed acute HIV infection or primary HIV infection) often have a negative test result.

What Abnormal Results Mean

A positive result on the ELISA screening test does not necessarily mean that the person has HIV infection. Certain conditions may lead to a false positive result, such as Lyme disease, syphilis, and lupus.

A positive ELISA test is always followed by a Western blot test. A positive Western blot confirms an HIV infection. A negative Western blot test means the ELISA test was a false positive test. The Western blot test can also be unclear, in which case more testing is done.

Negative tests do not rule out HIV infection. There is a period of time (called the "window period") between HIV infection and the appearance of anti-HIV antibodies that can be measured.

If a person might have acute or primary HIV infection, and is in the "window period," a negative HIV ELISA and Western blot will not rule out HIV infection. More tests for HIV will need to be done.

Risks

Veins and arteries vary in size from one patient to another and from one side of the body to the other. Obtaining a blood sample from some people may be more difficult than from others.

Other risks associated with having blood drawn are slight but may include:

Excessive bleeding
Fainting or feeling light-headed
Hematoma (blood accumulating under the skin)
Infection (a slight risk any time the skin is broken)

Considerations

People who are at high risk (men who have sex with men, injection drug users and their sexual partners, commercial sex workers) should be regularly tested for HIV.

If the health care provider suspects early acute HIV infection, other tests (such as HIV viral load) will be needed to confirm this diagnosis, because the HIV ELISA/Western blot test will often be negative during this window period.

Alternative Names

HIV testing

References

Dewar R, Goldstein D, Maldarelli F. Diagnosis of human immunodeficiency virus infection. In: Mandell GL, Bennett GE, Dolin R, eds. Principles and Practice of Infectious Diseases. 7th ed. Philadelphia, Pa: Elsevier Churchill Livingstone; 2009:chap 119.

Sax PE, Walker BD. Immunopathogenesis of human immunodeficiency infection. In: Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia, PA: Saunders Elsevier; 2007:chap 408.

Western Blot: Technique, Theory, and Trouble Shooting

Abstract

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize. This paper will attempt to explain the technique and theory behind western blot, and offer some ways to troubleshoot.

Keywords: Bio-medical research, protein, western blot

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Introduction

Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest.

The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present. The paper will first describe the protocol for western blot, accompanied by pictures to help the reader and theory to rationalize the protocol. This will be followed by the theoretical explanation of the procedure, and in the later section, troubleshooting tips for common problems.

Technique

Cell lysis to extract protein

Protein can be extracted from different kind of samples, such as tissue or cells. Below is the protocol to extract proteins from adherent cells.

Adherent cells:

Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout).

Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.

Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.

Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail).

Incubate for 30 minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.

Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C.

Measure the concentration of protein using a spectrophotometer.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/

Western Blot Procedure

Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.

1) Proteins are separated by gel electrophoresis, usually SDS-PAGE.

2) The proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. 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. 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.

4) 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.

http://www.bio.davidson.edu/courses/genomics/method/westernblot.html

Overview of Western Blot Hybridization

The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Western blotting (also called immunoblotting because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Western blotting can produce qualitative and semiquantitative data about that protein.

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Video: Probe the blot for your target proteins

Detection Methods

Modified protocol for Western blotting

A. Preparation of cell lysates

1. Harvest cells (70-85% confluent) by trypsinization and spin.

2. Lyse the pellet with 100 µl lysis buffer on ice for 10 min.

Note: 1) Just scrub the cells after washing with PBS and adding the lysis buffer, if the cells being plated at dishes or plates.

2) Sonication is optional after lysing the sample, but just don’t sonicate the sample too long. We suggest it should be sonicated for no more than 10 seconds.

3) The amount of lysis buffer is flexible. It depends on the number of cells you have.

4) You need to homogenize it first if you do Western blotting with tissue.

1. Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 20 min at 4°C.

2. Transfer the supernatant to a new tube and discard the pellet.

3. Measure the protein concentration with protein assay whichever method set up by your lab. (e.g. Bradford assay, or BCA; or Lowry Assay)

Note: Don’t dilute the protein too much. You can concentrate the protein lysate with vacuum under 4ºC in case you got a very low protein concentration.

1. Take the same amount protein lysate from each sample separately, and mix with sample loading buffer. The final concentration of loading buffer must be 1X.

e.g. The ratio of sample (ul)/ 2X loading buffer (ul) should be 1:1. The ratio of sample (ul)/ 6X loading buffer (ul) should be 5:1.

1. Boil for 5 min.

2. Cool at room temperature (RT) for 5 min.

3. Briefly spin to bring down the sample and loading buffer mixture prior to loading gel.

refer to: http://www.e-biotek.com/protein/244-western-blot-protocol.html