Biochem Lab Midterm 1

The closer the R² value is to 1.0, the better the linear regression fits your data points, and the more precise and accurate you’re pipetting (T/F)

True

Name the pipette part: A

Plunger

Name the pipette part: B

Friction Ring

Name the pipette part: C

Micrometer

Name the pipette part: D

Volumeter

Name the pipette part: E

Shaft

Name the pipette part: F

Tip Ejector Button

Name the pipette part: G

Body

Name the pipette part: H

Tip Ejector

Name the pipette part: I

Pipette Tip

Gel filtration chromatography

Also known as size exclusion chromatography (SEC), When a mixture of molecules is dissolved in a liquid and then applied to a chromatography column that contains porous beads, large molecules pass quickly around the beads, whereas smaller molecules enter the tiny holes in the beads and pass through the column more slowly.

Affinity Chromatography

a biomolecule (often an antibody) that will bind to the protein to be purified is attached to the beads. A mixture of proteins is added to the column and everything passes through except the protein of interest, which binds to the antibody and is retained on the solid support. To get the protein to elute from the column, another buffer is used to disrupt the bond between the protein of interest and the antibody. Often this elution buffer contains high concentrations of salt or acid.

Ion Exchange Chromatography

the glass beads of the column have a charge on them (either + or −). A mixture of protein is added to the column and everything passes through except the protein of interest

Anion Exchange

If the charge of the beads is positive, it will bind negatively charged molecules

Cation Exchange

If the beads are negatively charged, they bind positively charged molecules

Beads (SEC)

Microscopic porous spheres that as has traps to filter small molecules that are temporarily trapped within the pores, also known as the stationary phase

Mixture of molecules dissolved in liquid (SEC)

The mobile phase

Column Bed (SEC)

The mass of beads within a column

Exclusion Limit (SEC)

Molecules greater than 60,000 pass around the beads and are excluded from the columns

The smaller the molecules, the slower they move through the column in size exclusion chromatography (T/F)

True

Buffer (SEC)

The liquid used to dissolve the biomolecules to make the mobile phase

Sample (SEC)

The mixture of biomolecules dissolved in the buffer

Identify the type of chromatography described by each statement.

A positively charged bead interacts with the protein to be purified, which carries a negative charge. A high salt buffer is used to elute the protein to be purified.

Ion-Exchange Chromatography

Identify the type of chromatography described by each statement.

Beads are linked to a biomolecule that interacts with the protein to be purified. The protein to be purified is sometimes eluted using a buffer containing a high concentration of salt or acid.

Affinity Chromatography

Identify the type of chromatography described by each statement.

Beads containing small pores are used to separate biomolecules based on their size. Large biomolecules have little interaction with the beads and elute first, whereas small biomolecules have a lot of interaction with the beads and elute later.

Size Exclusion Chromatography

Identify the type of chromatography described by each statement.

A negatively charged bead interacts with the protein to be purified, which carries a positive charge. A high salt buffer is used to elute the protein to be purified.

Ion-exchange chromatography

Imagine you have a mixture containing two proteins, untagged green fluorescent protein (GFP) and His-tagged GFP (His-GFP). Which type of chromatography would be useful to separate these proteins?

Affinity Chromatography

What type of chromatography is being used in this lab?

Size Exclusion chromatography to separate haemoglobin and vitamin B12

How does the type of chromatography being used in this lab function?

proteins are separated by…?

Size

What gives hemoglobin and myoglobin their distinctive colors?

Iron-containing heme

If athletes wanted to increase their endurance by increasing their oxygen-carrying capacity, how might they accomplish this increase?

Training at high Altitudes

How could gene-therapy, where a normal gene is substituted for a defective copy of a gene, help individuals who have vitamin B12 carrier protein disease?

By providing a working copy of the gene

Range of P1000

100-1000uL

Tip colour of P1000

Blue

Range of P200

20-200 uL

Tip colour of P200

Yellow

Range of P20

2-20 uL

Tip colour of P20

Clear

A micropipette is used to pipette specific, small volumes. What is the volume pipetted if the dial reads the following for a P200?

47uL

A micropipette is used to pipette specific, small volumes. What is the volume pipetted if the dial reads the following for a P1000?

470uL

A micropipette is used to pipette specific, small volumes. What is the volume pipetted if the dial reads the following for a P20?

4.7uL

If a P20 micropipette has the pictured setting, what volume of liquid will be dispensed?

14.6uL

If a P200 micropipette has the pictured setting, what volume of liquid will be dispensed?

72uL

If a P1000 micropipette has the pictured setting, what volume of liquid will be dispensed?

530uL

Target A

Precise and Accurate

Target B

Precise but not Accurate

Target C

Accurate but not precise

a protein found in red blood cells, functions to transport oxygen to the tissues of the body

Hemoglobin

The body can not adapt to environmental changes which require increased amounts of oxygen delivery to tissues (TF)

False

Immunology

study of the immune system

Innate immunity

A person is born with certain immunological defenses against pathogens, includes circulating macrophages and natural killer cells

Passive immunity

the acquisition of antibodies from an external source, for example, antibodies passed from mother to infant, or certain postexposure vaccines such as that for rabies.

Passive immunity lasts only a few weeks, and also does not change with multiple exposures (T/F)

True

Acquired/Adaptive immunity

Specific response to specific foreign substances, split into 2categorties: humoral immunity and cell-mediated immunity

Humoral Immunity

involves production of antibodies that circulate in the bloodstream and lymph and bind specifically to foreign antigens

Cell-mediated immunity

involves the production of T lymphocytes (T cells) that bind and destroy infected cells

Antigen

In an immune response, an invasion by something foreign to the body generates antibody production by B lymphocytes (B cell)

Epitope

Each B lymphocyte generates a unique antibody that recognizes a single shape on an antigen

Antigens

can be microorganisms (e.g., viruses and bacteria), microbial products (e.g., toxins produced by some bacteria, or protein components of the microbes), foreign proteins, DNA and RNA molecules, drugs, and other chemicals

Antibodies

Proteins also called immunoglobulins (Ig), that are produced by B cells and can remain attached to B cells or become free floating

5 classes of immunoglobulins

IgG, IgM, IgA, Ig E, and IgD

IgE is the most abundant immunoglobulin (T/F)

False, IgG is the most abundant 15% (parham)

Immune cells

soldiers of the acquired immune response

Macrophages two primary functions

1) Removing foreign cells and molecules from the blood / 2) Processing antigens and presenting them on their cell surfaces

Disease can result from… (3)

Infection, genetic defect, or environmental toxins

Infections can be…

Transmitted from person to person, Transmitted from animals to people, or contracted from the environment

Pathogen Spread Through: Exchange of body fluids

Sexually transmitted diseases

Pathogen Spread Through: Food

Mad cow disease

Pathogen Spread Through: Water

Cholera and Giardiasis

Pathogen Spread Through: Inhalation

Flu and Tuberculosis

Pathogen Spread Through: Absorption through the skin

Hookworms

Pathogen Spread Through: Vector transfer

Malaria

Primary Response

When immunized with a foreign substance (either by vaccination or through natural exposure), an individual mounts an immune response

Secondary Response

The second time that the individual is exposed to the antigen, be it weeks or years after immunization, the immune response is larger and much more rapid

Antibody production may continue for a couple days or weeks (T/F)

False, months or even years

Problems with the immune system (3)

Hypersensitivity, Immunodeficiency, and autoimmune diseases

Hypersensitive Reactions

occur when the immune system overreacts to an antigen

Four types of hypersensitive reactions

1) anaphylactic reactions, allergies

2) Cytotoxic Reaction, transfusion reactions

3) Immune complex reactions, Farmer’s lung

4) Delayed-type hypersensitivity, Dermatitis

Immunodeficiency

an individual is unable to mount an effective immune response, resulting in increased vulnerability to opportunistic infections

Two types of immunodeficiency

1) Genetic basis (“bubble boy” disease)

2) External cause (HIV/AIDS)

Autoimmune disease

results from the immune system making a mistake and mounting an immune response against one’s own body

Immunofluorescence assay (IFA)

Specific microorganisms detected with fluorescently labeled antibodies

Agglutination

Visible precipitates appear when antibodies and specific antigens come in contact

Immunochromatography tests

Card or dipstick-based immunoassays

Microplate tests

ELISA or RIA

Molecular methods

Detection of microbial RNA or DNA; also used to detect microbial drug resistance (AST); may use PCR

Microscopy

Visual identification based on staining with specific reagents or on physical characteristics

Live attenuated vaccines

are weakened (attenuated) microbes, that are non-pathogenic. Ex. Measles and smallpox

Killed or inactivated vaccines

are made of microbes killed by heat or chemicals. Ex. Rabies

Subunit Vaccines

are made from pieces of microbes. They consist of one or more antigens from either the disease agent or a microbial product, and they may be derived from the organisms or engineered using molecular biology. Ex. Tetanus

DNA Vaccines

DNA that codes for microbial antigens is cloned into a vector, and the naked DNA is injected into the patient. The DNA is taken up by cells, transcribed, and translated, and the resulting antigenic protein elicits an immune response. Not available yet.

mRNA Vaccines

The molecules are made of mRNA bases which carry chemical modifications that increase the mRNA's stability and immunogenicity. The mRNA molecules are taken up by cells and translated to produce the antigen, which is then used to produce an immune response. Ex. COVID-19

Antibody Vaccines

The ability to construct human monoclonal antibodies using recombinant DNA technology means that antibodies prepared against specific antigens may be used safely in humans.

Postexposure Vaccines (immunotherapy)

are used to treat a disease

Two types of traditionally produced antibodies

Polyclonal Antibodies and Monoclonal Antibodies

Polyclonal antibodies

generated by immunizing an animal (usually a rabbit, goat, or sheep) and obtaining serum. The product is antiserum towards gp120, and the antiserum can be used directly or the antibodies can be purified from it.

Advantage/Disadvantage of Polyclonal antibodies

Simple, inexpensive but no batches will be the same

Monoclonal Antibodies

B cell clones producing single antibodies can be isolated from the spleens of immunized mice, but these cells die after a few weeks in the laboratory, limiting production of the large amounts of antibody generally needed for research and commercial applications. However, B cells can be made to live (and produce antibodies) indefinitely if they are fused with tumor-like immortal cells

Genetic engineering antibody methods (2)

Hybridoma Immortalization and Phage Display

Hybridoma Immortalization

Recombinant DNA technology allows the antigen recognition site from a known mouse monoclonal antibody to be camouflaged within a human antibody by combining part of the mouse gene with the human antibody gene