Lab #2

Question 1

How do cells typically respond to stress factors like moderate temperature fluctuations at a molecular level?

Answer 1

They utilize protective mechanisms by up-regulating or down-regulating protein expression.

Question 2

What specific cell line is being used in this experiment?

Answer 2

CHO (Chinese Hamster Ovary) cells.

Question 3

What are the three temperature conditions used for incubation in this experiment?

Answer 3

4C, 37C, and 44C.

Question 4

What is the first major procedural step after collecting the incubated CHO cells?

Answer 4

Extracting the proteins from the cells.

Question 5

What is the primary purpose of performing a Bradford Assay in this lab?

Answer 5

To accurately determine the protein concentration within the extracted samples.

Question 6

According to the learning outcomes, how is the protein concentration of the unknown samples determined from the Bradford Assay data?

Answer 6

By constructing a standard curve.

Question 7

What future techniques (Labs 3 and 4) require the protein concentration data obtained today?

Answer 7

SDS-PAGE and Western Blotting.

Question 8

In the previous lab, what variable was tested regarding CHO cell viability at -20C?

Answer 8

The presence or absence of DMSO during cryopreservation.

Question 9

What are the four main learning outcomes for this lab?

Answer 9

1. Demonstrate pipetting proficiency.
2. Describe protein extraction.
3. Understand Bradford Assay theory.
4. Construct a standard curve.

Question 10

What is the fundamental structure of the cell biology experiments described?

Answer 10

Testing the effect of a single variable on a measurable phenotype.

Question 11

What are three examples of ‘measurable phenotypes’ mentioned in the text?

Answer 11

1. Regulation of protein expression (up or down).
2. Subcellular location of a protein.
3. Functionality of a protein.

Question 12

What is the first step in setting up these experiments, prior to counting?

Answer 12

Growing large quantities of cells.

Question 13

After collecting cells in a tube, what measurement must be taken before dilution can occur?

Answer 13

Determining the concentration of the collected cells.

Question 14

What is the specific target concentration mentioned for seeding cells in this procedure?

Answer 14

1 times 10^5 cells/ml.

Question 15

What is the final step in the setup procedure after the cells have been diluted?

Answer 15

Seeding the cells into vessels to start the experiment.

Question 16

In the context of this procedure, what is the purpose of the dilution step?

Answer 16

To ensure the cells reach the specific final concentration required for standardized seeding.

Question 17

What is the specific variable being tested in this multi-week experiment?

Answer 17

Temperature stress (Heat shock or Cold shock).

Question 18

What term describes the sample that is exposed to the variable?

Answer 18

The ‘Treatment’ or ‘Experimental’ sample.

Question 19

What is the definition of a ‘Negative Control’?

Answer 19

A sample not exposed to the variable that serves as a baseline to compare against.

Question 20

Why is a Negative Control necessary?

Answer 20

To ensure that observed results are caused by the variable and not by other unknown factors.

Question 21

How should a Negative Control be handled procedurally?

Answer 21

Identically to the experimental sample in every way, except for the variable.

Question 22

What specific condition constitutes the ‘Cold Shock Treatment’ (C)?

Answer 22

Incubation at 4C.

Question 23

What specific condition constitutes the ‘Heat Shock Treatment’ (H)?

Answer 23

Incubation at 44C.

Question 24

What specific condition constitutes the ‘Negative Control’ (N) in this experiment?

Answer 24

Incubation at 37C (the standard physiological temperature).

Question 25

Why is the 37C sample considered the Negative Control here?

Answer 25

Because it represents the absence of the 'stress' variable.

Question 26

What is the general goal of protein extraction?

Answer 26

To release proteins from cells by disrupting membranes and collecting the supernatant (cell lysate).

Question 27

What are the two main categories of approaches used to extract proteins?

Answer 27

Physical methods and Chemical methods.

Question 28

How does Liquid Homogenization (e.g., Dounce homogenizer) work?

Answer 28

It shears cells by forcing them through a narrow space using a plunger and vessel.

Question 29

How does Sonication work to extract proteins?

Answer 29

It uses pulsed, high-frequency sound waves delivered via a vibrating probe to agitate and lyse cells.

Question 30

How does Manual Grinding work and what sample type is it best for?

Answer 30

Freezing tissue in liquid nitrogen and smashing it; it works well for plant cells.

Question 31

What are the two main disadvantages of physical extraction methods?

Answer 31

1. They are often inconsistent/less reproducible. 2. They generate heat, which can denature proteins.

Question 32

How do chemical extraction methods disrupt the cell?

Answer 32

They use detergents and/or hypotonic (low salt) solutions to disrupt the phospholipid membrane.

Question 33

What specific lysis buffer is used in today's lab?

Answer 33

RIPA buffer.

Question 34

What is the specific detergent component found in RIPA buffer?

Answer 34

Triton X-100.

Question 35

Why are protease and phosphatase inhibitors included in the RIPA buffer?

Answer 35

To prevent the extracted proteins from degrading or denaturing.

Question 36

What is the main objective of Task #1 in this lab session?

Answer 36

To lyse the CHO cells and extract their proteins.

Question 37

During the cell collection phase, why are the old media, PBS wash, and trypsinized cells all transferred into the same Falcon tube?

Answer 37

To ensure every single cell is collected for pelleting, rather than discarding those floating in the old media.

Question 38

What is the function of Trypsin in this procedure?

Answer 38

It is an enzyme used to detach the adherent cells from the bottom of the flask.

Question 39

Why is fresh media added and 'aggressively mixed' after the trypsin incubation?

Answer 39

To neutralize the trypsin and mechanically break up cell clumps.

Question 40

After the first centrifugation (500 rpm), where are the cells located?

Answer 40

In the pellet at the bottom of the tube (the supernatant is discarded).

Question 41

What reagent is added to the cell pellet to perform the actual lysis?

Answer 41

RIPA buffer.

Question 42

Why must the lysate sit on ice for 12 minutes after adding RIPA buffer?

Answer 42

To allow time for chemical lysis to occur while keeping the sample cold to prevent protein degradation.

Question 43

After the second centrifugation (in the microcentrifuge), what separates into the pellet vs. the supernatant?

Answer 43

The pellet contains cell debris (broken membranes); the supernatant contains the extracted proteins.

Question 44

What is the specific purpose of the tube labeled 'B'?

Answer 44

It holds the protein lysate that will be used immediately for the Bradford Assay.

Question 45

What is added to the 'Storage' tube before it is placed in the freezer for Lab 3?

Answer 45

Sample Buffer (the blue liquid).

Question 46

What is the primary purpose of a Bradford Assay?

Answer 46

To determine the total concentration of protein in a solution.

Question 47

What visual change occurs when the Bradford reagent binds to protein?

Answer 47

The color changes from brown to blue.

Question 48

How is the intensity of the reaction (the 'blueness') quantified?

Answer 48

By measuring absorbance using a spectrophotometer set at 595 nm.

Question 49

What acts as the 'known' standard in this experiment?

Answer 49

BSA (Bovine Serum Albumin).

Question 50

What relationship does the 'Standard Curve' demonstrate?

Answer 50

A positive, linear relationship between protein concentration and absorbance.

Question 51

How do you mathematically determine the concentration of your unknown sample?

Answer 51

By using the equation of the line generated from the BSA standard curve.

Question 52

Why must you prepare two different dilutions (10x and 20x) of your unknown CHO lysate?

Answer 52

To ensure that at least one absorbance reading falls within the linear range of the standard curve.

Question 53

What does the notation '[protein]' represent?

Answer 53

The concentration of protein, typically measured in ug/ul.

Question 54

What specific samples are prepared in cuvettes 1 through 4?

Answer 54

Duplicates of a 20x dilution (Trial #1 & #2) and duplicates of a 10x dilution (Trial #1 & #2).

Question 55

What is the composition of the 'Blank' cuvette?

Answer 55

It contains only PIPES buffer (no protein lysate).

Question 56

What is the purpose of the 'Blank' cuvette?

Answer 56

It serves as a zero reference point for the spectrophotometer to correct for background absorbance.

Question 57

What buffer is used to dilute the cell lysate in this specific step?

Answer 57

PIPES Buffer.

Question 58

What reagent is added to the cuvettes to initiate the reaction?

Answer 58

Bradford reagent.

Question 59

How should you mix the cuvettes after adding the Bradford reagent?

Answer 59

Seal with Parafilm and invert several times.

Question 60

How long must the reaction incubate before the color change is finalized?

Answer 60

10 minutes.

Question 61

What visual change indicates that protein is successfully present in your dilutions?

Answer 61

The solution turns a blue color.

Question 62

How does a spectrophotometer measure absorbance?

Answer 62

It uses a photocell to convert light energy passing through the sample into electrical energy.

Question 63

What is the relationship between absorbance and the solution in the cuvette?

Answer 63

Absorbance is directly related to the concentration of the absorbing substance (protein) in the solution.

Question 64

Why is a 'Blank' cuvette used to zero the machine?

Answer 64

To subtract the background absorbance of the brown Bradford reagent so that readings reflect only the protein-dye reaction.

Question 65

What does the 'Blank' cuvette contain?

Answer 65

PIPES buffer and Bradford reagent, but no protein.

Question 66

Do you need to re-zero the spectrophotometer between every sample reading?

Answer 66

No, you only zero it once with the Blank at the beginning.

Question 67

What wavelength is used to measure the Bradford Assay reaction?

Answer 67

595 nm.

Question 68

After recording your own data, what step is required regarding the other group at your bench?

Answer 68

You must share data so that every group has results for both a Treatment and a Negative Control.

Question 69

How are the glass cuvettes cleaned to ensure the blue stain is removed?

Answer 69

They are washed with methanol.

Question 70

Where should the T25 flasks and plastic pipettes be disposed of?

Answer 70

In the biological waste bucket.