SDS PAGE
What is SDS-PAGE used for
What is protein purification
What is western blotting used for
what is Protein identification
What is mass spec used for
what is protein identification
How fast does a small protein move relative to a large protein in Size-Exclusion chromatogrpahy?
the large protein moves faster than the small protein
Name 3 ways to lyse a cell
-Mechanical stress
-Osmotic pressure
-Chemical basis
What kind of bonds/interactions are SDS molecules breaking apart?
What are non-covalent interactions (hydrogen bonds, hydrophobic interactions, salt bridges...)
After which process does Western blotting follow
SDS page
What measurement do you get from mass spec
You have m/z, which can be converted to the MW of the fragment of protein.
Upon what property of a protein does R-HPLC rely on?
hydrophobicity
Which functional group is responsible for fluorescence at 280nm?
an aromatic ring
What is the role of BME in SDS Page
What is reducing disulfide bonds
What does the anitobody recognize on the protein of interest, how does it recognize it?
paratope of antibody recognizes epitope (linear sequence of amino acids)of the antigen(protein), by binding (non covalent interactions between epitope and paratope).
What factors contribute to the "Time of Flight" of a peptide fragment
Size and charge (small and more charged makes it to the detector faster)
In what process is a His-tag used for protein purification?
Affinity chromatography
What is an example of a fusion protein that allows visualisation of a POI
GFP (green fluorescent protein)
What step must be done is order to visualise the proteins after the gel has run
what is staining using coomasie blue
What is used for blocking? And what is the purpose of blocking
Milk or BSA is typically used. Blocking prevents antibodys from unspecifically binding to the membrane or other proteins.
Name a struggle with using mass spec to identify between certain amino acids.
Some amino acids have the same MW ex. I and L
Describe how the POI is released from the exchange resin in ion-exchange chromotography, and how POI is released from molecule/ ligand in affinity chromatography.
Ion-exchange: increasing salt concentration or changing the pH
Affinity chromatography: Using competitor (ex. imidizole for HIS tag)
What are two important consideration when lysing a cell?
the release of proteases (add protease inhibitors)
Denaturation of proteins from conditions (add buffers+ice)
Because it creates a uniform mass-to-charge ration that allows the proteins to be seperated based only on molecular weight
What produces the chemoilluminescent signal in the final step of western blotting?
The chemoilluminescene is a result of the enzyme attached to the secondary antibody converting a substate present in the final incubation.
Once you know the MW of your protein fragments, how can this information be used to sequence the protein?
Using a database with all known proteins, the MW of your fragment is compared to the combined MW of the amino acids that may build up the fragment.
what are the exchange resins used in Ion Exchange chromatography?
Cation exchange resin:Carboxymethyl groups (CM)
Anion exchange resin: Diethylaminoethyl (DEAE)
Describe how a fusion protein is formed