WESTERN BLOT LAB EXAM 2 ❤️

SDS PAGE: Understanding the role of SDS PAGE in protein analysis.
iBlot: Familiarizing with the iBlot method for protein transfer.
ELISA: Discussion will be divided into Part 1 (included in Exam II) and Part 2 (included in the final exam).

Determining the Presence of a Protein: To ascertain if the protein of interest is present and approximately how much of it is.
mRNA: Detecting mRNA does not guarantee that it has been translated into protein.
Protein Localization: Techniques such as FISH (Fluorescence In Situ Hybridization) are used to determine where within the cell the protein is located.
Functional Analysis: Investigating protein function can involve manipulating the gene at the genetic level.

Definition: Proteins are polymers made up of amino acids.
Amino Acids: Each amino acid consists of a central carbon atom, an amino group, a carboxyl group, and a distinctive side chain (R group) which determines its properties.
Charged Side Chains: The charge varies based on R group characteristics.
Functional Groups:
- Carboxyl group:
- Structure: (-COO-)
- Function: Can lose a proton, giving a negative charge.
- Amino group:
- Structure: (-NH3+)
- Function: Can accept a proton, giving a positive charge.
Amphoteric: Proteins can act as acids or bases, capable of donating or accepting protons depending on the pH.
Isoelectric Point (pI): The pH at which the protein has no net charge, meaning the positive and negative charges are balanced.

Cell Lysis: Important to think about the end goal to select appropriate lysis method.
Functional Studies: Avoid heat and detergents, rely on mechanical disruption.
Presence Analysis: Structure of the protein is less important; utilize protease inhibitor cocktail and ensure equipment is at 4°C.
Protein Quantification: Conduct a protein assay, typically colorimetric, using a standard to create a graph for extrapolation.

Steps:
1. Protein Separation: Achieved by gel electrophoresis.
2. Transfer: Protein is transferred from the gel to a membrane.
3. Development: Visualization of the membrane for protein detection.

Purpose: Separation of proteins based on size.
Gel Composition:
- Key components: Polyacrylamide, ammonium persulfate, TEMED, and buffer.
- Monomer: Acrylamide is neurotoxic and operates as a mild conductor.
Buffer System: Includes Tris buffer, SDS, and 2-mercaptoethanol.
Sample Buffer: Contains glycerol, SDS, and dye, typically at a 1:2 dilution of the sample.
Parameters:
- Running Conditions: Apply a current of $150V$ for $90$ minutes.
- Molecular Weight Standards: Run parallel to test sample.

Structure:
- Front and Back Plates: Must enable proper gel function to load samples in wells.
- Wells to accommodate samples of DNA or protein.
- Considerations for differing single-strand DNA molecules used in comparisons.

pH of Running Buffer: Affects protein charge and migration rate based on isoelectric point (pI).
Charge Behavior:
- Acidic Proteins: Tend to accept protons, hence appear positively charged.
- Basic Proteins: Tend to donate protons and appear negatively charged.
SDS Effect: SDS imparts negative charges to proteins, affecting migration speed.

Staining Methods:
- Coomassie Brilliant Blue: Combined with acetic acid and methanol for protein fixation and staining.
- Silver Stain: More sensitive, good for detecting lower protein concentrations.
Destaining: Using acid-alcohol to reduce background staining.

Membrane Choice: Utilize nitrocellulose membranes due to their affinity for DNA, RNA, and protein molecules.
iBlot System: Facilitates dry transfer in a rapid timeframe of $7$ minutes.
Conventional Method: Involves wet transfer over $4-6$ hours.

Verification: Use a reversible stain (e.g., Ponceau Red, which stains the protein but not the membrane) to confirm successful transfer.
Blocking Process: Combines $5\%$ nonfat dry milk and $0.1\%$ Tween $20$ in PBS to reduce background interference from nonspecific antibody binding.

Primary Antibody: Example includes anti-BSA raised in rabbit, diluted with $2\%$ nonfat milk in PBS.
Washing Steps: Five washes with PBS + $0.1\%$ Tween $20$ to prevent high background.
Consequences of Inadequate Washing: Increased nonspecific binding leading to false positive signals.
Secondary Antibody: Goat anti-rabbit IgG conjugated with alkaline phosphatase for signal amplification.

Washing Steps: Additional five washes necessary post-secondary antibody binding.
Visualizing with Chromogenic Solution: Substrate NBT/BCIP reacts with alkaline phosphatase; enzyme activity leads to color change from colorless to purple indicative of protein presence.

Detection Sensitivity:
- A signal indicates presence; absence of a signal does not confirm absence of protein concentration, but suggests absence or undetectable levels.
- Quantitative Analysis: Semi-quantitative assessment can be achieved using internal controls like actin for normalization.

Polyclonal Antibodies: Generated from different plasma cells to target various epitopes.
Monoclonal Antibodies: Derived from a single clone, targeting a specific antigen epitopes.

Chemiluminescent Methods: In addition to chromogenic approaches, chemiluminescent substrates are used for light emission upon cleavage by enzymes, providing an alternative means for protein detection.