Principles of Biochemistry Midterm 1/2
Studied by 14 people
Call with Kai
Knowt Play
Learn
Practice Test
Spaced Repetition
Match
Flashcards1/82
Earn XP
Description and Tags
Contains information from LabFlow modules on Micropipetting and SEC Chromatography only. Second set will contain ELISA and Bradford Assay. Fill in the blank not recomended.
Name | Mastery | Learn | Test | Matching | Spaced |
|---|
No study sessions yet.
83 Terms
Why is proper micropipette use important?
Mistakes can cause failed or irreproducible experiments, delays, expense, and contamination.
What unit of volume do micropipettes measure?
Microliters (µL)
How big is a microliter?
About 40–50 µL = 1 drop of water.
What is accuracy?
How close measurements are to the true or expected value.
What is precision?
How close repeated measurements are to each other
What does an R² value indicate in pipetting?
How well data fits expected values; closer to 1.0 means higher precision and accuracy.
What type of pipettes are commonly used?
Air displacement pipettes.
What is the volume range of a P2 micropipette?
0.2–2 µL.
What is the volume range of a P10 micropipette?
1–10 µL.
What is the volume range of a P20 micropipette?
2–20 µL.
What is the volume range of a P100 micropipette?
20–100 µL.
What is the volume range of a P200 micropipette?
20–200 µL.
What is the volume range of a P1000 micropipette?
100–1000 µL.
What color are standard P1000 tips?
Blue.
What color are standard P200 tips?
Yellow
What color are P20 electrophoresis tips?
Clear.
What is the correct method to set a pipette volume?
Rotate volume wheel above desired value, then slowly turn down to set point.
How should pipette tips be attached?
Press pipette shaft gently into the tip with light force for a good seal.
What is the first stop on the plunger used for?
Aspirating liquid.
What is the second stop on the plunger used for?
Dispensing liquid completely.
Why should the pipette be held vertically (within 20°)?
To ensure correct aspiration without bubbles or error.
How should the tip be disposed of?
Use ejector button or dispose in biohazard/sharps container.
Why pre-wet the pipette tip 3 times?
To improve accuracy and reproducibility of volume delivery.
What happens if you let the plunger snap back?
It can damage the piston and cause inaccurate volumes.
Why should pipettes not be laid down with liquid inside?
Liquid can enter the piston and cause damage/contamination.
How often should pipettes be calibrated?
Every 6 months
How can you check for pipette leaks?
Fill tip with water, hold vertically for 20 sec; if a drop falls or level changes, there is a leak.
What should always be used to avoid cross-contamination?
A new pipette tip for every sample.
What is chromatography used for? :
To separate and purify biological molecules, like proteins, from complex mixtures.
What are the two phases of chromatography?
Mobile phase (buffer + sample molecules) and stationary phase (resin/beads in the column).
How does chromatography separate molecules?
Molecules move at different rates through the stationary phase based on properties.
What does size exclusion chromatography (SEC) separate by?
Size of molecules only.
How do large molecules behave in SEC?
Excluded from bead pores, move quickly, elute in early fractions.
How do small molecules behave in SEC?
Enter bead pores, move slowly, elute in later fractions.
What is the exclusion limit?
The molecular weight cutoff above which molecules cannot enter pores.
What molecules are separated in the SEC lab?
Hemoglobin (65,000 Da, brown) and Vitamin B12 (1,350 Da, pink).
Which molecule elutes first in the SEC lab?
Hemoglobin, because it is larger than the exclusion limit.
Which molecule elutes later in the SEC lab?
Vitamin B12, because it is small and enters pores.
What is hemoglobin’s function?
Transports oxygen in red blood cells.
What gives hemoglobin its color?
The iron-containing heme group.
What disease is caused by a mutation in hemoglobin?
Sickle cell anemia.
Why does sickle cell trait provide evolutionary advantage?
Protects against malaria infection.
What is Vitamin B12’s function?
Essential cofactor for biochemical reactions, including fat metabolism.
What foods are sources of Vitamin B12?
Animal products like eggs, dairy, and meat (not found in plants).
How is Vitamin B12 absorbed?
By binding to a carrier protein in the intestine before entering the bloodstream.
Why are vegetarians at risk for B12 deficiency?
Plant foods lack B12, so supplementation is needed.
What is collected from the SEC columns?
Fractions of the mobile phase containing separated molecules.
What protein was used in the SEC experiment?
Bovine hemoglobin (to study size-based separation)
What was the experimental procedure for SEC?
Pack column with porous beads → Equilibrate with buffer → Apply protein sample → Elute with buffer → Collect fractions → Analyze elution profile
What is the principle of affinity chromatography?
Separates proteins based on specific binding interactions (e.g., enzyme–substrate, antigen–antibody, His-tag–Ni²⁺)
What is the stationary phase in affinity chromatography?
Beads with covalently attached ligand
How are bound proteins eluted in affinity chromatography?
By changing conditions such as pH, salt concentration, or adding a competitive ligand
What is the advantage of affinity chromatography?
Very high specificity and purification power
What is the principle of ion exchange chromatography?
Separates proteins based on charge differences
What are the two types of ion exchangers?
Cation exchanger (binds + proteins) and Anion exchanger (binds – proteins)
How are proteins eluted in ion exchange chromatography?
By increasing salt concentration or changing pH
What does SEC separate proteins by?
Size/shape
What does Affinity Chromatography separate proteins by?
Specific biological interactions
What does Ion Exchange Chromatography separate proteins by?
Charge
What is a buffer in chromatography?
The liquid used to dissolve biomolecules (mobile phase) and carry them through the column
What does "fractionate" mean in chromatography?
To separate a mixture into individual components (fractions) based on size or other properties
What is the exclusion limit of a size exclusion column?
The molecular weight above which molecules are too large to enter the pores and are excluded from the beads (for this experiment, 60,000 daltons)
What is a column bed?
The mass of beads packed in a chromatography column through which the sample passes
What is the sample in SEC?
The mixture of biomolecules dissolved in buffer that is applied to the column
What does it mean when a molecule is "excluded" in SEC?
It is too large to enter the pores of the beads, so it passes quickly through the column
What is the purpose of collecting multiple fractions in the SEC?
To separate large and small molecules into distinct tubes for analysis
In ion-exchange chromatography, how does a positively charged bead interact with a protein?
It binds to proteins that carry a negative charge
In ion-exchange chromatography, how is the protein eluted from the column?
By using a high salt buffer to disrupt the interaction between the protein and the bead
In affinity chromatography, what is linked to the beads to purify a protein?
A biomolecule (often an antibody) that specifically interacts with the protein of interest
In affinity chromatography, how is the protein typically eluted?
By using a buffer containing a high concentration of salt or acid to disrupt the protein-biomolecule interaction
Which type of chromatography would be useful to separate untagged GFP from His-tagged GFP?
Affinity chromatography (using beads that specifically bind the His-tag)
How can athletes increase their oxygen-carrying capacity to improve endurance?
By training at high altitudes, which increases the number of red blood cells and hemoglobin
How could gene therapy help individuals with a defective vitamin B12 carrier protein?
By providing a working copy of the gene, allowing proper absorption of vitamin B12
Why does the column need to be "dry" (no buffer on top) when loading the protein mixture?
To prevent disturbing the column bed, ensure the protein mixture enters the beads evenly and allows proper separation.
Why do you need to add more buffer after the protein mixture is loaded onto the column?
To push the sample through the column, maintain flow, and allow the proteins to separate based on size as they pass through the beads