What was the specific purpose of using DMSO in Lab 1?
It acted as a cryoprotective agent to preserve CHO cell viability during freezing.
How were the CHO cells physically stressed in Lab 2?
They were subjected to temperature shock: either cold (4�C) or heat (44�C) for 1 hour.
What method was used to determine the protein concentration of the lysate?
A Bradford Assay.
What is the specific biological question being investigated regarding Hsp90 or tubulin?
Whether CHO cells increase or decrease expression of these proteins in response to temperature shock.
What is the primary method used to separate the CHO proteins in this lab?
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).
To ensure a valid comparison of expression levels what must be consistent when loading the gel?
You must load the same amount of total sample protein into each lane.
After protein separation on the gel what is the immediate next procedural step?
Transferring the proteins from the gel onto a piece of nitrocellulose membrane.
What is the purpose of transferring proteins to a membrane (Western Blotting) in Lab 4?
To assess if protein expression has changed between control and treated samples.
What are the two main learning outcomes for this specific lab session?
To describe protein gel electrophoresis theory and demonstrate pipetting proficiency.
What is the primary function of the “gel” matrix in electrophoresis?
It acts as a molecular sieve that allows molecules to migrate differentially based on size.
In terms of gel composition and separation basis how do Protein and DNA electrophoresis differ?
Proteins use Polyacrylamide (separate by molecular weight); DNA uses Agarose (separate by base pairs).
Why do smaller molecules migrate faster than larger molecules in the gel?
Small molecules fit easily through the pores; larger molecules become entangled in the matrix.
What are the two main functions of SDS (Sodium Dodecyl Sulfate) in the Sample Buffer?
- Disrupts protein folding (denaturing to rod-shapes). 2. Coats proteins with negative charges.
Why is it important that SDS masks the existing charges of the proteins?
To ensure all proteins are negatively charged so they migrate towards the positive electrode based only on size.
SDS disrupts protein folding but what component is required to break disulfide bonds?
A reducing agent (such as beta-mercaptoethanol or DTT).
What is the procedural purpose of boiling the samples for a few minutes before loading?
To ensure the proteins have fully denatured.
How does Native-PAGE differ from SDS-PAGE regarding the Sample Buffer?
Native-PAGE omits denaturing chemicals (SDS) and reducing agents to conserve protein structure and interactions.
What is the purpose of running Molecular Weight Standards (Ladders) alongside your samples?
To create a standard curve used to estimate the molecular weight of the sample proteins.
What is the practical advantage of using “pre-stained” markers?
They allow you to track the migration of proteins in real-time while the gel is running.
What are the four components previously mixed into the cell lysates via the Sample Buffer?
SDS
Why is it critical to calculate the specific volume required to load exactly 15 ?g of protein per lane?
To ensure a fair comparison where differences in band intensity are due to biological changes (expression) rather than loading errors.
Which specific sample tube must NOT be heated/boiled?
The Molecular Weight Standard (labeled “M”).
Why must the tubes be centrifuged briefly after heating?
To eliminate bubbles and bring the solution/condensation back to the bottom of the tube.
When preparing the pre-made gel cassette what three items must be removed before insertion?
The packaging
