Antibodies & Protein Analysis Methods

Importance of Protein Analysis - The Why

• To determine a protein’s function in a cell

  • What does it do and what is it important for

• Understanding a protein’s function can help understand:

  • Why certain diseases appear

  • Designing therapeutics or pharmaceuticals

• Understanding a protein’s function can extend to understanding:

  • How other molecules interact with each protein

  • What the main activity of the protein is (e.g., transport, signaling, etc.)

  • Protein regulation

  • Location of protein

  • Continuous vs. discontinuous expression

Importance of Protein Analysis - The When

• When we do not know or understand the function of a protein

• When we have some information about a protein, but need to understand more fully what the function of a protein is

  • Example: Knowing protein A interacts with protein B, but not knowing when and where this interaction occurs

Protein Analysis - How

• Focusing on techniques that utilize antibodies

  • Western blot

  • Immunofluorescence microscopy

  • Co-immunoprecipitation

Common First Step - Silico Analysis

• Function

  • Computational modeling/analysis of a protein

  • Requires that the sequence of the protein is known

• What different programs can PREDICT:

  • The secondary or tertiary structures of the protein

  • Protein domains that may be important for understanding the location or function of the overall protein

  • How the protein may interact with another molecule or protein

  • If the protein has been modified

    • Addition of sugar groups, phosphate groups, etc.

• IMPORTANCE

  • Helps provide initial information and insights into the protein

  • Helps pinpoint gaps in knowledge

  • Helps decide next steps, such as further appropriate protein analysis techniques that can be performed to bridge this gap in information

Antibody Structure

• Contains four variable regions

  • Two on light chain (shorter chain) and two on heavy chain (longer chain)

  • Definition: The part that varies in amino acid sequence between different antibodies

• Contains four constant regions

  • Two on light chain and Two on heavy chain

  • Definition: The part that remains the same in amino acid sequence between different antibodies of a type

• Disulfide bonds

  • Covalent bonds between cysteine molecules that help stabilize the tertiary and quaternary structure of antibodies

• Epitope

  • Part of antigen that allows the antibody to recognize and bind it

Antibody Production in Response to Infection

• B cells produce antibodies

  • As a result, there are different B cells for different antibodies

• Each B cell has multiple copies of the same membrane-bound antibody on it

  • This antibody is capable of recognizing one specific antigen

• When the specific antigen binds to the antibody, the B cells divide

  • Creation of an army of B cells

• The army of B cells will secrete the membrane-bound antibody into the stream and create more

• Alerts the biological system of an infection, which will lead to defense mechanisms that target the antigen

Raising Antibodies in Animals

• An antigen specific to the antibody of interest can be injected into a animal (usually a mouse, rabbit, sheep, or goat)

• Repeated injections of the antigen will stimulate B cells to secrete large amount of anti-A antibodies into the bloodstream

• NOTE: Since many different B cells can be stimulated by an antigen, it is possible for a variety of anti-A antibodies to be created

  • Each anti-A antibody will bind A in a slightly different way

Making A Cell Lysate

• Breaking open cells and tissues to extract protein

• Homogenization

  • A gentle mechanical procedure that causes the plasma membranes of cells to rupture so that cell contents (like organelles) are releases

  • Techniques

    • High-frequency found

    • Mild detergent that makes holes in the membrane

    • Forcing cells through a small hole using high pressure

    • Shearing cells between a close-fitting rotating plunger and the thick walls of a glass vessel

• Homogenate

  • “Thick soup”

  • Extract that contains large and small molecules from the cytosol including enzymes, ribosomes, organelles, etc.

SDS-PAGE

• Must be performed before a Western Blot can be conducted

• Procedure

  • Establish a fair playing ground for all proteins so that only their molecular weights are being compared

    • Heating up each sample of proteins (from one lysate) and adding SDS and Mercaptoethanol

      • Heating: denatures proteins so they go from their complex 3D shape to their linear polypeptide chain

      • SDS: Since each chain will inevitably have various charges, SDS coats each chain to make them all negatively charged

      • Mercaptoethanol: Helps break apart disulfide bonds between cysteine molecules that heat alone can’t

  • Each sample is loaded into its own well

    • A well will act as a marker that provides a reference to molecular weights

    • Since the samples are all negatively charged, they will travel down to the anode (positive side)

      • Small molecules travel further

• The resulting gel is further used in the Western Blot

• RESULTS

  • Can see size of protein

  • May be able to detect protein modifications

  • May be able to detect multimerization (quaternary structure

Western Blot

• After a SDS-PAGE experiment is conducted

  • Proteins will all be sorted by molecular weight BUT they will all be invisible

• In a Western Blot procedure

  • These sorted proteins are transferred onto a sturdy membrane (like paper)

    • Like a sandwich, the gel and membrane are pressed tightly together between filter paper and sponges

    • An electric field is applied perpendicular to the gel → proteins are pulled out of the gel and stick onto the surface of the membrane in exact same positions

  • PRIMARY ANTIBODY

    • Designed specifically to recognize the protein of interest

    • Will bind just to that protein in the field of thousands of other proteins

    • While it is very good at recognizing the specific protein, it is USUALLY INVISIBLE

  • SECONDARY ANTIBODY

    • Designed to BIND to the PRIMARY antibody at the CONSTANT region

      • Makes it so that secondary antibodies can be universally used for each type of antibody instead of specific antibodies

    • This antibody is chemically linked to an enzyme or fluorescent dye

      • Makes the location of the protein finally visible

• VISUALIZATION

  • Tells the relative abundance of the target protein by how dark/light the blot is

Immunofluorescence Microscopy

• NO CELL LYSATE

• Procedure

  • Cells are applied with a chemical in order to freeze the cells’ components in place so that the structure remains intact for further use

  • Cells are applied with a little bit of detergent to poke holes into the membrane so that antibodies will be able to travel through

  • Then, cells are places onto slides

  • Antibodies are added before everything is sealed up with the coverslip

    • Primary antibody for identification of protein

    • Secondary antibody to light up location of protein

  • Slides can be observed under microscope

• Helps understand how location of a target protein plays a role in the function they have

• Helps understand how proteins interact too

  • Using a green tag for protein A and a red tag for protein B → If you see a mix of these colors at a spot, then the proteins possibly interact

(Co)-Immunoprecipitation

• Used to identify protein-protein interactions

• USES A CELL LYSATE

• Procedure

  • A very gentle detergent is used for the cell lysate because it is important to not break any of the weak, noncovalent bonds that hold all the proteins together

  • A primary antibody specific to the “bait” protein is added to the sample and binds to the protein of interest

  • It is nearly impossible to pull out the antibody with the “bait” on it SO heavy microscopic beads coated in a protein are added

    • They bind to the CONSTANT region of the antibody

  • Now the chain is: Bead → Antibody → Target Protein → Any additionally attached proteins

  • For magnetic beads, place a tube next to a magnet and everything that’s not connected to the bead will wash away

  • Perform a Western Blot on the pile of beads with the target protein and its attached proteins

    • Blot to make sure the bait protein is actually captured

    • Blot for additional proteins