Biol 233 Final Study Guide

State Beer’s Law. Equation.

A=ε⋅c⋅l
Where:

• A = absorbance

• ε = molar absorptivity

• c = concentration of the solution

• l = path length of light through the solution. 

State Beer’s Law. Sentence.

Beer’s Law states that the absorbance of light by a solution is directly proportional to the concentration of the absorbing substance and the path length of the light through the solution.

What is a spectrophotometer? How does it work?

A spectrophotometer is an instrument that measures how much light a solution absorbs. It works by passing light of a specific wavelength through a sample and measuring the intensity of light before and after it passes through.

What wavelength is the spectrophotometer set to detect? Why?

Usually 280 nm for proteins (due to aromatic amino acids absorbing UV light).

This wavelength correlates with protein concentration.

Define proteome

All proteins expressed by a cell, tissue, or organism.

Define proteomics

Study of the structure, function, and interactions of all proteins in a system.

Describe myosin proteins in general

Motor proteins that interact with actin to generate force.

Describe myosin II in particular.

Found in muscle; forms thick filaments and powers muscle contraction

Explain how a sarcomere contracts.

Myosin heads bind actin → power stroke pulls actin toward center → sarcomere shortens → muscle contracts.

How were proteins extracted and denatured from fish muscle?

Proteins were extracted using a buffer that breaks open cells and solubilizes proteins, and denatured using SDS and heat, which unfolds the proteins and gives them a uniform negative charge

Why extract muscle proteins from different fish?

To compare myosin light chain sizes and properties across species

Different fish species were used to compare their protein amounts and properties, particularly to get enough protein for statistical comparisons like a t-test.

What is SDS PAGE? Why is it used?

SDS PAGE is a method to separate proteins by size using a polyacrylamide gel. SDS denatures proteins and gives them a uniform negative charge, so separation is based on size, not shape or charge.

List components for SDS PAGE and their functions.

Gel: matrix to separate proteins.

Buffer: maintains pH and conducts electricity.

SDS: denatures proteins and provides uniform charge.

Samples: proteins to be separated.

Micropipette: loads samples accurately.

Purpose of running stained standards:

To track protein migration and estimate the molecular weight of unknown proteins.

Rabbit actin/myosin as positive/negative control:

The rabbit sample confirms the detection system works (positive) and shows specificity, meaning it should only detect the proteins it’s supposed to (negative).

How did you know when to stop electrophoresis?

Tracking dye reached bottom of gel → proteins fully separated

What is Western blotting? Why is it used?

Western blotting transfers proteins from a gel to a membrane for detection using antibodies, allowing identification of specific proteins.

Draw, label, give functions of blotting sandwich. What causes protein movement?

Sandwich: Gel Holder Cassette→ Sponge → Filter paper → PAGE gel → Membrane → Filter Paper → Sponge → Gel Holder Cassette

Movement: Electric field moves negatively charged proteins onto membrane

The blotting sandwich is layered to keep the membrane wet and in contact with the gel and filter papers, ensuring efficient transfer of proteins from the gel to the membrane.

What is immunodetection? Examples?

Using antibodies to detect specific proteins.

Examples: Western blot, ELISA, immunofluorescence

Primary antibodies & how made:

Bind directly to target protein (antigen)

Made by injecting antigen into animal → immune response → collect serum

Secondary antibodies & how made:

Bind to primary antibody

Made by injecting antibodies from one species into another species → immune system produces antibodies against them

Purpose of HRP (horseradish peroxidase) & location:

Enzyme conjugated to secondary antibody

Produces color/chemiluminescence when substrate added

Located on secondary antibody

How does secondary antibody help detect small amounts of antigen?

Multiple secondary antibodies bind one primary → amplifies signal

Purpose of blocker milk solution:

Blocks non-specific binding sites on membrane → reduces background

How did you collect data to make a standard curve?

By measuring band intensity and comparing it to standards of known concentration or molecular weight.

Two methods to estimate MW; which is more accurate?

Methods: Compare migration on gel, use standard curve

More accurate: Standard curve

What does R² indicate?

How well data fits the regression line; closer to 1 = better fit

What exactly did you plot for standard curve? Why?

Migration distance (or relative mobility) versus log of molecular weight, to allow accurate estimation of unknown protein sizes.

How did you convert estimate into kD?

By using the standard curve equation to calculate the molecular weight in kilodaltons

Why different species have same proteins but different MW:

Isoforms, post-translational modifications, slight sequence differences

Understand how myosin works with actin.

Myosin binds to actin filaments and, using energy from ATP hydrolysis, pulls the filaments to shorten the sarcomere, causing muscle contraction.

What is SDS?

SDS (sodium dodecyl sulfate) is a detergent that denatures proteins and coats them with a negative charge.

Understand the math to do the standard curve.

Use the linear relationship between migration distance and log(MW), plug in your protein’s migration distance, and solve for MW.

What are the 3 Kaleidoscope Standards in SDS-PAGE?

Pre-stained protein markers in the ladder used as reference points to track protein migration and estimate molecular weights. They are usually three prominent, colored proteins (e.g., ~250 kD blue, ~100 kD green, ~50 kD red) that are easy to see during electrophoresis.

Why are Kaleidoscope Standards used in SDS-PAGE?

They allow you to track gel progress, visualize multiple proteins at once, and estimate the sizes of unknown proteins.

What does “each step is for a reason” mean in SDS-PAGE and Western blotting?

Every step in the workflow has a functional purpose, such as:

• Protein extraction & denaturation: solubilize and unfold proteins

• Loading dye: denature proteins and provide tracking dye

• Electrophoresis: separate proteins by size

• Stained standards: track migration & estimate size

• Transfer/blotting: move proteins to membrane

• Blocking: prevent nonspecific antibody binding

• Primary antibody: bind target protein

• Secondary antibody + HRP: amplify signal and produce color change