L3: SDS-PAGE, Isoelectric Focusing, 2D gel electrophoresis, Probe

What type of technique can be conducted to reveal how much protein was in an original sample from a mixed protein sample

Protein Assay

What type of protein gel can be done on a sample of different proteins in cells?

SDS-PAGE

• more proteins = more bands in gel

What is done to prepare a protein sample?

• cells are ground up to release proteins

• Using Fluorescent Revealers or a Tag to bring the protein of interest to the surface and hide the other proteins we are not interested in.

How is a protein of interest identified?

Antibody is required to detect the specific protein, revealed using a Western Blot

What does an SDS-PAGE protein gel do?

Separates proteins based on size/molecular weight

What type of gel is used in SDS-PAGE?

Polyacrylamide Gel

What is Isoelectric Focusing?

Technique that separates proteins based on differences in their Isoelectric Point (pI), which is related to charge

What is the Isoelectric point (pI)?

The pH at which a protein or amino acid has NO NET Electrical Charge and is least soluble

• allowing it to precipitate out of solution

What type of gel is used in Isoelectric focusing?

Polyacrylamide Gel

What happens to an amino acid when it is dissolved in water

The amino acid takes an ionic form as water breaks covalent bonds, creating H+ and OH- ions

Draw a generic amino acid

What happens to amino acids in BASIC conditions? (NaOH)

The amino group stays the same, while the carboxyl group loses H+ to form water, leaving oxygen negatively charged.

What are the charges in a Polyacrylamide gel during Isoelectric focusing?

• Acidic pH (1-6) near the positive electrode

• Basic pH (8-14) near the negative electrode

How are two amino acids linked together

Peptide bond

• releasing H2O as a byproduct

What happens if an amino acid that is basic is loaded at the pH 7 well in the Polyacrylamide gel and is NOT at its Isoelectric point?

It will migrate toward the negative electrode because of a positive charge on the nitrogen

• there would be OH- groups floating around more than H+ because of the solution migrating to basic above pH 7

• When pH increases, OH- increases resulting in a H+ leaving the nitrogen to make it a neutral charge

What happens to a protein when it reaches its Isoelectric point in a gel?

It becomes electrically NEUTRAL and stops migrating

How does the Isoelectric point technology help separate proteins

Based on where they achieve a neutral charge, by adding or removing protons

If 3 bands appear at pH 4, 5, and 9 on a Polyacrylamide gel, how many proteins are in the sample?

3 proteins, each sharing the same pI value.

What is SDS-PAGE used for after Isoelectric focusing?

To separate proteins based on molecular weight

• a single band can contain proteins with the same pi but different molecular weight

What are the steps of 2D gel electrophoresis

1. Run Isoelectric focusing gel (IEF) with Polyacrylamide gel

2. Soak the IEF gel into SDS

3. Load the IEF gel as a source of proteins for an SDS-PAGE

4. Run SDS-PAGE

Why is IEF run with a Polyacrylamide gel during 2D gel electrophoresis?

To allow the proteins to separate based on pI

In an SDS-PAGE how do proteins migrate on the gel?

Based on molecular weight

How do proteins migrate on an SDS-PAGE gel with the Polyacrylamide gel strip ?

Based on Isoelectric points (pI)

How is data represented in 2D gel electrophoresis

Each protein is represented by a spot, not a band

What does a spot on the SDS-PAGE gel indicate

Proteins with the same pI but different Molecular Weight will form separate sport

What is the purpose of a kDa ladder in SDS-PAGE

It helps estimate the molecular weight of proteins

True/false: a single band can have multiple proteins that share the same molecular weight

True

A 2D gel electrophoresis is a technique used to separate proteins based on what two things?

1. Molecular weight (MW)

2. Isoelectric point (pI)

How is molecular weight separated on a 2D gel electrophoresis

Separated by SDS-PAGE based on size

How is pI separated on 2D gel electrophoresis?

By Isoelectric focusing gel which separated proteins based on their pI

What is a way you can study ONE PROTEIN you are specifically interested in from a mixture?

Using a Probe

What is a Probe?

Tool used to help identify, tag, or highlight a protein of interest

What are the two functions of a Probe

1. Bind specifically to your interest

2. Way to reveal itself by using chemical reactions such as:

   • fluorescent signal

   • Radioactive activity

   • Enzymatic reaction

How can a probe tag your protein of interest

By obtaining a chemical that can identify its specific shape

What can create a good foundation of creating a good probe to identify a protein?

Antibodies

When you get sick or injured, your body defends itself by sparking your immune system to create what 3 things?

1. Antibodies

2. T-cell activity

3. Inflammation

What are antibodies (immunoglobulins)?

• Y-shaped proteins made of 4 polypeptides (2 long chains, 2 short chains) held together by disulfide bridges

• They have a quaternary structure

• Can be cell bound or free floating

What are the two regions of an antibody?

1. Hyper variable regions

2. Constant regions

What are the constant regions on an antibody

Similar across antibodies of the same class

How are antibodies made?

Made by B-cells

How are B-cells made?

Produced in the bone marrow

What do B-cells create?

Antibodies with unique hyper variable regions to target specific antigens

How do B cells differ?

Each B cell makes identical antibodies, with a specific hyper variable regions that differs from other B cells

What determines the effectiveness of B cells?

The more diverse the hyper variable regions, the broader spectrum of antigens the immune system can detect

What holds antibody chains together

Disulfide bridges connect the long and short chains

When a foreign protein is present from a virus or bacteria, the hyper variable region of it can interact with other epitopes of a foreign protein. What happens with the B cells when this interaction occurs?

The B cells will replicate (clonal expansion)

• there are chemical and genetic signals that undergo mitosis to create many identical cells of the same type of antibody to fight the foreign protein

What is an advantage of the immune system

We can use animals that create immune cells to create antibodies of a particular protein

What happens when a rat is injected with your protein of interest?

It will create antibodies and B-cells

• a booster shot is then added to make more B cells to create antibodies that will specifically bind to the protein of interest at a particular epitope