Western Blotting

Overview of Western Blotting

  • Powerful technique used to detect specific proteins or their modifications in complex samples.

    • Samples: whole cell lysates, tissue samples, biological fluids, whole organisms, proteins from interaction assays, purified proteins.

  • Evolution to "Quantitative Western" methods that assess amounts of target proteins rather than just presence/absence.

Western Blot Protocol

  1. Sample preparation and quantification.

  2. Electrophoresis for protein separation.

  3. Transfer proteins to a blotting membrane.

  4. Blocking to prevent non-specific interactions.

  5. Incubate with primary antibodies and wash.

  6. Incubate with secondary antibodies and wash again.

  7. Detection, imaging, and analysis.

  • Optimization required at each step for reliable results.

Sample Preparation

  • Goal: disrupt cells/tissues to release soluble proteins while avoiding degradation, modifications, or precipitation.

  • Key practices:

    • Extract proteins quickly, on ice, using buffers with appropriate pH and ionic strength.

    • Include protease inhibitors to prevent degradation.

Electrophoresis

  • Essential for protein separation based on molecular weight with high resolution.

  • Typically uses denaturing systems, including:

    • SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) with different buffer systems: Laemli, Bis Tris, Tris acetate.

Electrophoretic Transfer

  • Blotting Method: Transfer proteins from gel to solid support membranes (nitrocellulose or PVDF).

    • Proteins retain charge (usually negative due to SDS) and are fixed at their gel migrating positions.

    • Moved out of gel toward membrane by electrical force, a little bit of charge is required.

Blotting Membranes

  • Options include nitrocellulose and PVDF. = porous

  • Pore size ranges between 0.1 and 0.45 μm.

  • Protein binding through non-covalent bonds and hydrophobic interactions

  • Sensitivity depends on amount of protein on membrane and accessibility to ABs.

Blotting Types

  • Wet Transfer:

    • Gel and membrane immersed in transfer buffer

    • Current applied across gel toward membrane, requires cooling.

    • Transfer is slow but higher efficiency and quality for large proteins.

  • Semidry Transfer:

    • Faster, less buffer used, but lower efficiency especially for large proteins

    • Membrane in direct contact with gel and filter paper = sandwich —> placed between electrode plates. Proteins move toward membrane and anode (+)

    • Requires careful management (voltage and time) to avoid overheating and sample loss due to high voltage gradient.

Transfer Buffer Variations

  • Different buffers are formulated based on:

    • Type of transfer (wet or semidry), membrane type, protein molecular weight.

    • Most contain methanol: strips SDS from proteins to ensure efficient binding to membrane BUT shrinks pores of gel = prevents efficient transfer of large proteins

    • SDS: excess must be removed from gels by equilibration in transfer buffer (10-30min)

      • May decrease binding of protein to membrane (in particular NC not PVDF)

      • BUT complete stripping may prevent protein transfer to membrane, especially large proteins

      • Require low concentrations 0.01-0.05% SDS in transfer buffer

  • Common buffers:

    • Towbin: 25mM Tris, 192mM glycine, 10-20% methanol, pH 8.3. Wet and semi-dry

    • Bjerrum and Schafer-Nielsen: 48mM Tris, 39mM glycine. Semi-dry

    • CAPS: 10mM CAPS, pH 11, 10% methanol; used for large proteins and downstream sequencing

    • Dunn carbonate: 10mM NaHCO3, 3mM Na2CO3, pH 9.9, 20% methanol; may produce higher efficiency transfer and improve antibodies binding

Blocking Step

  • Block sites on membrane to prevent non-specific antibody binding, which can yield high background signals.

  • May cause decrease of specific signal by competing with AB for specific epitopes

  • Preliminary testing of range of blocking reagent REQUIRED to ensure low background and no loss of signal

  • Various reagents:

  • Blocking reagents = high concentration of proteins in buffers with/without detergents

    • Common: BSA (1-5%) —> IgG cause high background

    • non-fat dry milk —> not used for phosphoproteins, endogenous Biotin may interfere with biotin-streptavidin detection

    • commercial blocking agents.

Antibody Basics

Immunoglobulin G: AB = immunoglobulin proteins synthesized in response to foreign substance (antigen)

  • Primary Antibodies: Bind specifically to target proteins, produced in response to antigens.

  • Secondary Antibodies: Bind to primary antibodies, often labeled for detection (e.g., conjugated with enzymes or fluorescent dyes).

    • Always commercial. Commercially covalently bound to enzymes, fluorescent dyes, biotin

    • Can be cross-absorbed with immunoglobulins from species evolutionary close to primary AB to achieve minimal cross-reaction with them.

  • Types of antibodies:

    • Polyclonal: Recognize multiple epitopes on single protein, usually higher affinity, lower specificity.

    • Monoclonal: Specific to single epitope, higher specificity but lower affinity.

  • Choice of labeled antibodies:

    • Enzymes: alkaline phosphatase (AP) and horseradish peroxidase (HRP)

    • Fluorescent dyes

    • Biotin

    • IMPORTANT: labeled primary AB are sometimes used in WB to improve specificity but detection with them is less sensitive.

Detection Methods

  1. Colorimetric Detection: Generates colored insoluble products at reaction sites (e.g., substrates for HRP and AP).

  2. Chemiluminescence Detection: Light emitted from enzymatic reactions (conversion of substrate to product); very fast and sensitive. Good documentation, allows multiple exposures for best image wide dynamic range

  3. Fluorescence Detection: Antibodies labeled with fluorophores; allows multiplexing but lower relative sensitivity than chemiluminescence, more expensive.

Imaging

  • Visible light scanners: after chemiluminescence detection and blots stained with visible light dyes.

  • CCD cameras:used with trans-illumination by light boxes after chromogenic/fluorescence detection

  • Laser and diode array scanners: highest sensitivity, resolution, linear dynamic range

Quantitative Western Blotting (QWB)

  • Aims to measure the amount of protein relative to a control (e.g., comparing treated vs. untreated). E.g. pull down experiment.

  • Requirements include:

    • Confirmation that signal intensity proportional to amount of loaded protein.

    • Proteins and loading controls quantified within linear range.

    • Normalizing signals against internal loading controls (e.g., housekeeping proteins).

    • Low variability to maintain reproducibility.

  • Dynamic range = range of intensities that detection instrument can measure in a single capture

  • Linear range = range of signal intensities recorded by detector that shows a linear relationship with amount of protein

    • high sensitivity without saturation is essential for a wide linear dynamic range

Normalization Techniques

  • Corrects for unavoidable sample-to-sample and lane-to-lane variations by comparing signals from target protein and an internal control in sample.

  • If no normalization: don’t know if changes in detected band intensities of target protein reflect biological change or variability in sample preparation and transfer.

    • Loading equal amount of protein —> not sufficient b/c loading inconsistencies and transfer variability.

Loading control requirements

  • Appropriate internal loading control: endogenous proteins used to indicate asmple concentration

  • Loading control must be unaffected by experimental conditions

  • Must be detected with same linear range as target

  • Does not interfere with target detection

Total protein as internal loading control (ILC)

  • Robust and reliable assessment = “golden standard”

  • After transfer but before blocking. Membrane is stained for protein —> proteins per sample lane are quantified and used fro normalization. Error and variability minimized due to integrated signal

  • Shows inconsistencies of transferring process

Housekeeping proteins as internal loading control

  • Assumed stable level of expression in variety of normal and treated samples. BUT some not stable in cells under different treatments.

  • Critical to verify expression is constant

Quantitative Analysis

- QWB results expressed as ratios (e.g., fold change and percentage change to reflect relative abundance).

Technical replicas = repeated measurements to establish degree of variability of protocol. Establish precision and reproducibility of assay.

Biological replicates = measurements of biologically independent samples to determine if experimental effect is biologically relevant.

Fold Change=normalized signalsamplenormalized signalcontrol\text{Fold Change} = \frac{\text{normalized signalsample}}{\text{normalized signalcontrol}}

  • Relative abundance as percentage change calculated as: (Fold change -1) * 100%