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Enzyme-Linked Immunosorbent Assay

Measures antibodies, antigens, proteins, and glycoproteins in a biological sample

Diagnosis, pregnancy tests, test for contamination, allergens, disease progression

ELISA - INDIRECT

Step 1: antibody Coating

Specific capture antibody is immobilized on high protein-binding plates

Step 2: Capture

Samples and standard dilutions are added to wells and will be captured by bound antibodies

Step 3: Det4ectionAntibody

Specific detection antibody is added to enable detection of captured protein

If protein is absent, this antibody will attach to nothing

Step 4: Detection conjugated(enzyme)

Binds to detection antibody (like a sign)

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An enzyme called Cas9(produced by the CRISPR system) will bind to the matching DNA using a “spacer”

Spacer: Short RNA sequences that guide the CRISPR system to the target DNA

Cas9 will cut the DNA, shutting the targeted gene off

Research suggests CRISPR-Cas9 can be used to target and modify “typos” (aka mutations) in the human genome

There are other enzymes than Cas9 used in a CRISPR system

Ex. - Cpf1

PROS

Scientists can accerlerate research

Rapid diagnostic

Correct mutations

CONS

Ethics (how far is too far?)

Potentially wiping out species, creating bacterial resitance

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- Maintains homeostasis by controlling what goes in and what goes out
- Phospholipid bilayer
-Heads of lipids: Hydrophilic polar
-Tails of lipids: Hydrophobic nonpolar

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Movement from low to high concentration

Moves Against the gradient

Requires ENERGY

ATP!

ATP energizes the protein channel to transport molecule against the concentration gradient

Endocytosis(3 different types)

A type of active transport where the substance fuses with the cell membrane and brings the substance inside the cell in a vesicle

1. Phagocytosis

Used by amoebas to engulf a substance

2. ReceptorEndocytosis

Special receptor proteins capture a specific target molecule

3. Pinocytosis

Intake of liquid

Exocytosisis another form of active transport

Cell expels/releases/removes material OUT of the cell

This can include releasing neurotransmitters or proteins

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Molecules move from high to low concentration

Moves WITH the concentration gradient

Simple Diffusion

Very small nonpolar molecules can pass through the phospholipid with no help or energy

Molecules move from high to low concentration

Moves WITH the concentration gradient

Facilitated Diffusion

Uses a transport protein because molecule may be too big or polar

Doesn’t require energy

May require a stimulus (signal, usually an ion)

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Separates DNA by size

Shorter DNA travels further through gel

Utilizes Agarose

Agarose is very porous

Needs a buffer

This allows the electric current to travel through the gel

Uses:

Crime scenes

Paternal/Maternal testing

Comparing species

Identifying/observing certain diseases

When separating DNA, it all has the same charge

DNA will move from negative to positive ends!

DNA Ladder:

Known DNA sizes that are used as a standard to compare other samples

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How does it work?

Each line on the gel is a band of DNA.

Thicker bands mean there was a lot of DNA in that specific band

DNA samples that have similar banding patterns will be more closely related

Closely related organisms have similar DNA, therefore their banding patterns will also be similar

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Denuturation

Heatthe strands of DNA to break (denature) them apart into two separate strands

This gives room to replicate the desiredgene

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Polymerase Chain Reaction

Used to make MANY copies of a DNA region to study

3 main steps:

Denaturation

Annealing

Extension

PCR lets us take a smallsection of DNA and make hundreds of copies

This small section can be amplified and examined through many processes

For example, gel electrophoresis

Commonly used in

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Annearling

Cool DNA so primers can bind

Primers bind to areas of the DNA that we want to amplify

Two primers needed for each side of DNA

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Extension

Heat up DNA again to use taqPolymerase

Taq Polymerase adds nucleotidesto extend the primers and replicate DNA

This enzyme is adapted to work in high temps!

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Very similar process to the Western Blot, but this test looks for RNA, not protein

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Also known at Immunoblot

proteins

Detect protein of interest

Size of proteins

Amount of protein expressed

We’re using a charged solution to draw proteins through a gel

Proteins are sorted by mass and ability to bind to antiboides
Antibodies are Y-shaped proteins that bind to a specific target (antigen)

Used by our immune system!

How It Works

Proteins in the sample are coated with a detergent to denature (unfold) them AND give them a negative charge

We need linear proteins to move through gel

Take completed gel & membrane and sandwich between filter paper

Negatively charged proteins will be drawn to the positive end

The membrane is between the positive end of the chamber and the gel, so the proteins will be drawn onto the membrane

They’ll be “blotted” onto the membrane!

This membrane is an identical copy of the gel

Why the membrane?

Gel is too fragile for next steps

Antibodies could become stuck in the gel and give false results

Once proteins have moved onto the membrane, we add antibodies to locate the proteins we want to see

The antibodies act like a flag on the target protein

Once the membrane copies the gel, it’s moved to a chamber and exposed to primary antibodies

This primary antibody specifically attaches to the protein we’re looking for

Once attached, the membrane is washedso extra antibodies don’t stick around

A secondary antibody is then added to the membrane

This secondary antibody attaches to the primary antibody, not the protein

Contains a reporter enzyme that will produce light or color

The reporter enzyme allows us to locate the proteins!

By adding the antibodies, we only see the proteins we’re looking for on the final membrane