L7 Protein Purification Flashcards
(24 cards)
What does 6x His tag allow
purification of the GFP protein from other bacterial proteins
Histidine residues bind to _ ions including nickel, cobalt, and copper under specific buffer cond
metal
Affinity purification
Beaded agarose or magnetic particles with chelating groups can immobilize metal ions, which functions as ligands for binding and purification of 6xHis-tagged proteins
What buffer conditions are best for the binding of Ni-agarose to 6xHis tag?
near-neutral 7.2
Low concentration of imidazole helps to prevent
nonspecific binding of endogenous proteins that have histidine clusters
Imidazole is an ____ in the binding buffer
advantage
Why is it good to have imidazole in the binding buffer?
To remove contaminants while allowing His-tag protein binding
In affinity protein purification of His-tagged protein, why would you elute with imidazole?
To compete with the His-tagged protein for binding to the Ni-agarose beads
What is an advantage of size and shape gel filtration?
conditions can be varied to suit the sample or requirements for further processing
Good for molecules who may be sensitive to pH, ions, or harsh environmental conditions
Ion exchange chromatography IEX
Reversible interactions b/w charged molecules and oppositely charged IEX media are controlled in order to favor binding or elution of specific molecules and achieve separation
Due to Tyr and Trp residues, proteins have a characteristic absorption at
275-280nm
What impact could DNA have on the measurement of protein
We could overestimate [DNA] b/c DNA also contains aromatic rings
Bradford assay involves binding of ___ to ___
Coomasie blue to proteins
Under acidic cond the dye is__
red
When the dye binds the protein it is converted to a ___ form
blue
The blue protein-dye is detected at ___nm in the assay using a ____
595nm, spectrophotometer
What is the ideal way to select a protein standard?
A purified prep of the protein you are assaying - absolute reference std
If you cannot use a purified prep you can
use a relative standard which would need to be experimentally determined
What are the 4 protein assays
Enzyme activity
Binding assay
Immunological
Bioassay
Enzyme activity
measuring how much of a protein is required to digest a standard amount of substrate
Binding assay
Receptors might be assayed by its ability to bind ligands, which could be labeled by radioactivity or fluorescence
Immunological technique
Antibodies with highly specific affinity to certain proteins can be used in techniques to detect immobilized portiens
Bioassays
To detect specific biological acitivty
If acrylamide is less concentrated, there is ___ cross-linking and the more ___ a given protein migrates
less, rapidly
