Lecture 4 SDS PAGE and Western

Introduction to Protein Analysis Lab

Overview of Lab:

• The lab focuses on essential techniques such as SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), Western blotting, and the application of antibodies in protein detection and analysis. These methods are crucial for the study of protein size, structure, expression, and interactions.

• The lab begins with the quantification of proteins obtained from previous experiments, establishing a foundation for subsequent analysis.

Quantification of Protein Samples

Standard Curve Data:

• A standard curve was created using known concentrations of BSA (Bovine Serum Albumin), a standard protein used for calibrating protein assays.

• Absorbance values were measured at specific dilutions to generate reliable data, aiding in the estimation of protein concentrations in unknown samples.

Data Analysis:

• A scatter plot was constructed, plotting absorbance against concentration to visualize the relationship and identify trends.

• It was noted that a plateau in absorbance occurred at higher protein concentrations, indicative of dye saturation, which can obscure accurate measurements. To preserve the integrity of data, an outlier at 2 mg/mL was removed from the analysis, sustaining linearity in the graph.

• Trend lines were added with their intercepts set to zero to improve the statistical parameters, resulting in a high R-squared value of 0.9904, indicating a strong correlation between the absorbance and protein concentration.

Calculating Unknown Sample Concentrations:

• The absorbance readings for unknown samples were measured as follows: S1: 1.1 and P1: 0.6.

• By applying the established linear equation derived from the standard curve, the concentrations for the unknown samples were calculated: S1 was found to be 0.66 mg/mL, and P1 was determined to be 0.36 mg/mL.

• These concentrations were then converted into micrograms per microliter, resulting in S1 being 0.66 µg/µL and P1 being 0.36 µg/µL.

Loading Calculations:

• The lab aimed to load 50 µg of each protein sample onto the gel for analysis:

  • To achieve this, it was calculated that S1 required a volume of 76 µL to reach the 50 µg target.

  • For P1, a larger volume of 139 µL was required to meet the same mass target, thereby highlighting the need for careful volume planning due to loading volume limitations of 50 µL per well during gel electrophoresis.

Using Loading Dye:

• A four-fold concentrated loading dye must be diluted to reach a working concentration of 1X. The volume needed for 50 µL was computed using the formula: [ V1 (dye) = C2 (1X) * V2 (50 µL) / C1 (4X) ]

• This calculation resulted in needing 12.5 µL of loading dye, with the remaining 37.5 µL allocated for the protein sample mix.

Preparing Samples for Gel Loading:

P1 Sample Dilution:

• Based on the established concentration of 0.36 µg/µL and targeting a total volume of 50 µL, multiply concentration by maximum volume for desired protein content:

  • This calculated 13.5 µg of protein.

• The final mixture preparation for P1 consisted of: 37.5 µL of P1 combined with 12.5 µL of loading dye, together totaling 50 µL.

S1 Sample Calculation:

• Using P1’s reference concentration of 13.5 µg to maintain equality in protein loading, the required volume of S1 was calculated:

  • With S1 at 0.66 µg/µL concentration, the necessary volume was calculated as follows: [ 13.5 µg / 0.66 µg/µL = 20.5 µL of S1 ].

  • To complete the 50 µL mix: 17 µL of water was added to 20.5 µL of S1 and 12.5 µL of loading dye, totaling the desired mixture volume.

Understanding Polyacrylamide Gel Electrophoresis (PAGE)

Composition of Gel:

• The gel is comprised of acrylamide and bis-acrylamide polymerized under the influence of ammonium persulfate and TEMED, forming a porous matrix for protein separation.

• It is essential to handle acrylamide with care due to its neurotoxic properties, emphasizing safety precautions in the lab environment.

Gel Percentages and Pore Sizes:

• Gel percentages are critical in determining the pore sizes suitable for protein sizes:

  • 8% gel: optimal for proteins between 4-200 kDa.

  • 10% gel: suitable for proteins within 20-100 kDa.

  • 12% gel: designed for proteins ranging from 10-40 kDa.

Gel Structural Arrangement:

• Gels are to be loaded with a stacking gel (around 4%) on top of a resolving gel (4%-15% gradient). This structural design facilitates proper protein separation based on size during electrophoresis.

SDS PAGE Process

Role of SDS:

• Sodium Dodecyl Sulfate (SDS) imparts a uniform negative charge to the proteins, ensuring their separation primarily based on size rather than charge during electrophoresis.

• Proteins will undergo denaturation through heating post-loading dye incorporation, which is crucial for accurate separation.

Western Blotting Overview

Concept of Blotting:

• Originally developed by Edwin Southern for DNA analysis, the concept of blotting has been adapted for RNA (Northern blot) and proteins (Western blot). The current focus in the lab is on Western blotting techniques appropriate for protein identification.

Blotting Materials:

• Nitrocellulose membranes are employed for efficient transfer and immobilization of separated proteins.

• It is vital to ensure minimal loss of proteins, preventing them from adhering to the bottom filter paper during transfer.

Antibodies in Protein Detection

Structure of Antibodies:

• Antibodies consist of four polypeptide chains organized into two light chains and two heavy chains, forming a Y-shaped structure that plays a crucial role in specific antigen recognition.

• The structure contains variable regions (FAB) necessary for antigen binding and constant regions (Fc) that mediate interactions with other components of the immune system.

Antibody Production:

• Antibodies are produced by B cells in animals (e.g., rabbits, mice) upon antigen injection, triggering an immune response that leads to the development of polyclonal or monoclonal antibodies.

Polyclonal vs. Monoclonal:

• Polyclonal Antibodies: These recognize multiple epitopes on an antigen, providing greater variability but reduced specificity.

• Monoclonal Antibodies: These offer high specificity by binding to a single epitope and are produced from hybridoma cells, which are derived from spleen cells fused with myeloma cells.

Antigen Labeling Techniques:

• Antigens can be labeled directly or indirectly for signal detection.

• Secondary antibodies are often utilized for signal amplification and flexibility, with examples including fluorophore-conjugated secondary antibodies and enzyme-linked secondary antibodies (e.g., alkaline phosphatase).

Conclusion of Lab Preparation

• A summary of preparation requirements and methodologies for SDS PAGE and Western blotting is highlighted, emphasizing the importance of safety precautions when handling acrylamide.

• The anticipated outcomes from the lab experiments include the successful identification of the protein of interest through specific antibody binding, facilitating a deeper understanding of protein function and interaction within biological systems.