Western Blot Notes

Immunoblots

  • Immunoblots separate multiple antigens by electrophoresis to detect serum antibodies.

  • Multiple protein antigens are isolated, denatured, and separated using SDS-PAGE.

  • SDS denatures the protein and adds a negative charge proportional to the protein's molecular weight.

Western Blot

  • PAGE of SDS-treated proteins separates proteins by molecular weight.

  • Separated proteins are transferred to a medium, fixed, and stained.

  • Each lane is incubated with a patient sample, where the antibody binds to the antigen.

  • After washing, a labeled antibody detects the complex.

Blotting Techniques

  • Southern Blot: Target DNA, Probes: DNA or RNA sequence; Alternatives: PCR, real-time PCR, FISH

  • Northern Blot: Target RNA, Probes: DNA or RNA sequence; Alternatives: RT-PCR, real-time RT-PCR

  • Western Blot: Target Protein, Probes: Monoclonal antibody; Alternatives: ELISA, immunohistochemistry, immunofluorescence, flow cytometry

Blotting Protocols

  • Southern and Northern blots detect DNA and RNA via:

    • Electrophoretic separation of nucleic acid

    • Transfer to a solid support

    • Hybridization with a labeled probe

    • Autoradiographic or colorimetric detection

Western Blotting (Immunoblot)

  • Western blotting analyzes individual proteins in a mixture using gel electrophoresis (SDS-PAGE, etc.).

  • Separated proteins are transferred to a membrane (nitrocellulose, nylon, or PVDF).

Blotting Process

  • Proteins adhere to the membrane in a separated pattern due to charge interactions.

  • These proteins are accessible for antibody binding and detection.

Western Blot Technique

  • Western blot: Separates proteins electrophoretically, transfers to membranes, and identifies using labeled antibodies.

  • Used to detect antibodies to specific epitopes of separated antigens.

Western Blot Setup

  • Components: Tank, gel cassette, anode, cathode, buffer, power source.

  • Separates protein bands.

  • Diagram illustrates setup with gel, transfer membrane, filter paper, pads, support grid, and buffer.

Western Blot Protocol

  • Sample preparation: Lysis, protein assay, reduction, denaturation, and addition of sample buffer. Heat at 95°C for 5 minutes.

  • Loading the gel: Optimize lysate amount.

  • Running the gel: Apply 100V - 200V; optimize time and voltage. Gel percentage depends on protein size.

  • Smaller proteins move faster.

Protein Transfer Protocol

  • Transfer proteins: Prepare transfer buffer, cut membrane, assemble transfer stack.

  • Check the transfer: Use Ponceau red or Coomassie staining.

  • Blocking: Incubate the membrane in blocking buffer.

Antibody Incubation and Detection

  • Primary antibody incubation: Incubate with primary antibody for 1-2 hours at RT or overnight at 4°C.

  • Secondary antibody incubation: Incubate with secondary antibody for 1-3 hours at RT.

  • Detection: Use a substrate, then scan and analyze results.

Protein Transfer in WB

  • Components: Positive plate, sponge, filter papers, blotting membrane, gel, negative plate.

  • Wet Transfer

  • The PAGE gel is sandwiched between two membranes, filter paper, and glass plates, and incubated at 37°C for varying periods of time to obtain up to 12 blots.

Western Blot Specificity

  • Blotting separated antigen to nitrocellulose and reacting with patient specimen binds specific antibodies.

  • Electrophoresis of standards determines molecular weight.

  • Detected using EIA reactions.

  • Used to confirm antibody specificity detected by ELISA.

Western Blot HIV Analysis

  • HIV protein antigens are separated by electrophoresis and blotted onto strips.

  • Incubated with patient antibody, washed, and reacted with enzyme-conjugated antihuman antibody.

  • Serum from an HIV-infected person binds and identifies major antigenic proteins.

Detection

  • Diagram illustrates detection in Western Blots.

Detection signals

  • Yields photometric, fluorescent, or chemiluminescent bands.

  • Bands from patient sample are compared with controls.

  • Molecular weight markers provide a guide.

Why Western Blotting?

  • Western blotting expands on ELISA by separating the protein mix by size, charge, and/or conformation.

  • Allows detection of several targets and determination of protein size and content.

Limitations of WB

  • Time-consuming, requires expertise, and needs optimized conditions.

  • Specificity and sensitivity can be altered.

  • Not as accurate for quantification as newer techniques.

  • Destroys tertiary structure, affecting epitope recognition.