Western Blot Theory and Practice

Introduction to Western Blot Technique

• The video introduces the theory and practice of the Western blot technique, a common method for protein preparation.

• SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is highlighted as a method used for various purposes, including estimating molecular weight and assessing protein abundance in samples.

Gel Electrophoresis Setup

• A discontinuous gel system is typically employed, consisting of a separating gel and a stacking gel that improves resolution.

• SDS denatures proteins, imparting a uniform negative charge, which allows for separation based on molecular weight during electrophoresis.

Protein Migration Dynamics

• The migration speed of proteins in the gel is influenced by their relative molecular mass; larger molecules migrate slower than smaller ones.

• The relationship between migration distance (Rf value) and molecular weight follows an inverse logarithmic correlation.

Visualization Techniques Post-Electrophoresis

• After electrophoresis, protein bands are visualized using staining methods such as Coomassie Brilliant Blue or silver nitrate.

• Unlike direct staining methods, Western blotting involves transferring proteins from the gel to a membrane for further analysis.

Transfer Process to Membrane

• Proteins are transferred to membranes like nitrocellulose or PVDF using an electric current after electrophoretic separation.

• This transfer process allows for gene expression analysis of specific proteins in various tissues.

Antibody Incubation Steps

• Once on the membrane, proteins undergo sequential incubation with blocking agents and primary/secondary antibodies.

• Secondary antibodies often carry enzymes that facilitate detection through enzymatic reactions or fluorescence.

Practical Application: Evaluating Protein Expression

Preparing Samples for Western Blot

• The practical segment focuses on evaluating the presence of a specific protein (referred to as Protein X).

• Prior to running the gel, total protein concentration must be determined using methods like Bradford assay to ensure equal loading across samples.

Execution of Western Blot Procedure

Western Blotting Procedure Overview

Preparation of Gel for Western Blot

• The process begins with the assembly of a polyacrylamide gel for protein separation based on mass, requiring cleaning of glass plates with alcohol.

• A short plate and a 0.75 or 1.5 mm thick spacer are positioned to create a cavity for the gel, secured in place with a holder and rubber at the base.

• Two types of gels are prepared: stacking (concentrator) and separating gels; specific volumes depend on desired resolution and number of gels run simultaneously.

Gel Polymerization Steps

• Acrylamide is added last to avoid premature polymerization; once mixed, it must be poured into the mold immediately.

• A P1000 pipette is used to fill the mold without creating bubbles, followed by adding saturated isobutanol to facilitate even polymerization.

• After approximately 10 minutes at room temperature, isobutanol is removed, and any residue cleaned off without disturbing the gel.

Concentrator Gel Preparation

• For the concentrator gel (5%), components are also added last; it fills from the top while ensuring no bubbles form during placement of combs.

• The comb remains in place during polymerization for about 10 minutes to form wells for sample loading.

Sample Loading and Electrophoresis

• Samples consist of equal amounts (40 µg each), mixed with loading buffer containing SDS and dye for visualization during electrophoresis.

• Samples undergo heat treatment at 100°C for denaturation before being loaded alongside a molecular weight marker into wells created by the comb.

Transfer Process Post-Electrophoresis

• Following electrophoresis at constant voltage (150 volts), proteins are transferred onto a nitrocellulose membrane using specific materials soaked in transfer buffer.

• The transfer occurs from negative to positive pole at 80 volts over two hours on ice; verification steps follow to ensure successful transfer.

Verification and Blocking Steps

• Membrane rinsing in TBS-T followed by staining helps confirm proper protein transfer before proceeding further.

• If results appear satisfactory, membranes can be stored wet in TBS-T until continuation; otherwise, blocking prevents non-specific binding sites from interfering with antibody detection.

Incubation Phase

Western Blot Technique Overview

Antibody Usage in Western Blot

• The western blot technique employs two antibodies sequentially: a primary antibody that specifically recognizes the target protein (protein X) and a secondary antibody that binds to the primary, facilitating detection of the protein-antibody complex.

Key Factors for Successful Results

• Incubation of the membrane with antibody solutions is crucial for successful western blotting. This process typically uses a buffer solution containing hydrochloric acid and sodium chloride or phosphate-buffered saline (PBS).

• Parameters such as antibody dilution, incubation time, temperature, and washing duration are tailored to each antigen-antibody complex. Companies often provide optimized protocols with purchased antibodies.

Detection Methodology

• The most common detection system involves a secondary antibody covalently linked to horseradish peroxidase (HRP), which catalyzes luminol oxidation in the presence of hydrogen peroxide, producing light detectable by radiographic plates or chemiluminescence equipment.

Image Capture and Analysis

• Images of membranes can be captured using software like "snack" to determine the presence or absence of protein X by observing bands corresponding to each sample.

• Semi-quantification of bands is performed through densitometric analysis using software, normalizing against constitutive gene expressions like beta-actin or GAPDH from the same lane.

Quantification Insights

• The area under each curve corresponding to bands is quantified numerically and divided by values from control bands (beta-actin or GAPDH), yielding arbitrary units for relative expression comparison across samples.

• Examples illustrate results comparing steroidogenesis-related proteins in follicular samples from control rats versus those with surgically induced endometriosis during different estrous cycle phases.

Variability in Technique Application