Protein Techniques Flashcards
Insulin has ____side chains
2
To calculate MW you need to multiply the # of amino acids by ____
110
Hemoglobin has ____side chains
4
Hexokinase has ____side chains
2
RNA polymerase has ____side chains
5
What do conjugated proteins contain
prosthetic groups
What does protein quantification rely on
Absorbance characteristics of aromatic amino acids
Free amino group where
At amino terminus
What is ion exchange chromotography
Separates on basis of electric charge
Anion exchange column has what?
Resin has fixed cationic groups and binds anionic substances
What does cation exchange column depend on
Resin has fixed anionic group and binds cations
What is affinity chromatography
Based on binding specificity.
a. Resin is cross linked with a specific ligand
b. Protein to be purified will bind tightly
c. all other protein is washed away
d. Purified protein is released by addition of excess ligand
What is molecular sieve chromatography
Based on SIZE
a. Resin is comprised of beads with tiny pores
b. Large proteins cant enter beads and fall through column quickly
c. Small proteins are trapped and move slowly, while BIG things come out quickly
What is gel electrophoresis
Separates on basis of size
a. SDS imparts overwhelming negative charge on proteins
b. Proteins will migrate towards (+ pole)
c. Largest move slowly, smallest moves faster
What does SDS-PAGE stand for
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
What is Western blotting
Transfer to a membrane (paper filter) –>designed to bind proteins. Pushes protein out of gel and onto paper. Soak blot in specific antibody solution.
What is isolelectric focusing
Separates on basis of isolectric point
a. pH gradient is produced by allowing a mixture of ampholytes in PAGE to migrate to neutral positions in an electric field.
What is 2D gel electrophoresis
1st dimension: Isoelectric focusing
2nd dimension: SDS-PAGE
What is Proteomics
Uses 2D PAGE and Mass Spec to identify differences in protein profile of two cell populations
a. Shine light on cell; cells respond differently and start making different proteins based on light dosage
b. Label them with 2 diff dyes that will coalesce. Take two pops and mix them together and do 2D gel electrophoresis. When you expose them to infrared light you can see which ones are present in treatment group and not at all
What is Mass Spectrometry
a. Proteins are added to a light absorbing matrix, then pulsed with laser which ionizes the proteins
b. Proteins are desorbed from matrix and enter into vacuum.
c. Charged proteins introduced into magnetic fields
d. Path through field is a function of mass to charge ration
- Patterns are very specific!
What are antibody-antigen interactions
Identifies specific proteins using antibodies
What are the techniques that use antibody antigen interactions
- Immunoblots (Western Blots): Transfer of proteins from gel electrophoresis onto filter paper and soaking blot with antibody antigen solution
- ELISA: Plate is precoded with antibodies. Can put protein in solution. If protein your interested in is in solution it will bind to antibody and cause color change.
- Immunocytochemistry: Usually done with cells on culture. Using antibody to determine if a specific protein is present in tissue or cells that you’re working on.
What are antibodies
Proteins produced by B lymphocytes (immuglobulins) to destroy antigen containing foreign invaders.
What are antigens
Any kind of proteins that elecit an antibody response in your body.
