Protein Purification & Sequencing Flashcards
what is proteomics?
the study of protein
what are the two ways to determine the sequence of a protein ?
- Gene sequencing using dna sequence
- chemical and enzymatic sequencing, Mass spectrometry sequencing
when can separation techniques & monitoring protein [ ] and function be useful
- the function is known
- you dont know the protein sequence
- dont know size, charge
what are the 6 physico-chemical properties that seperation relies on ?
- size
- charge
- affinity for ligand
- solubility
- hydrophobicity
- thermal stability
what are the 2 ways to separate proteins based on the solublity
1.using ammonium slufate adding more salt to change ionic strengh allow for percipitation if the protein are very different therefore more soluble will stay longer but the will eventually come off at different rate
2. pH at pI
how can you separate by size ?
- using size exclusion chromotography where there are holes that the smaller one get stuck too and the largest ones pass through easily
what are the three exlusions limits of gel seperation
- 3000-15000
- 50,000-500,000
3.10,000-100,000
what happens to protein below the limit
the will elute “wash out” at the bed volume
what happens to the protein at greater than the limit
the will elute at the void volume
if we have exclusion range 30kd- 200KD in what order will they be seperated who is bed volume and who is void volume
myoglobin- 17 kd
hemoglobin- 68kd
imunoglobin - 150- 200kd
glutamine syntheaase- 400kd
1st void volume GS
2nd imo
3rd hemo
4th bed myo
if we have exclusion range 50kd- 500KD in what order will they be seperated who is bed volume and who is void volume
myoglobin- 17 kd
hemoglobin- 68kd
imunoglobin - 150- 200kd
glutamine syntheaase- 400kd
1st no void volume
2nd imnuo
3rd hemo
4th bed volume myo
what happens if the pH is below the pi? pH< pi
the protein is positvely charged
what happens if the pH is above the pi ? pH> pi
the protein is negatively charge
how can we seperate protein based on charge
using a postive or negative ion exchange resin
how do the postive and negative charge ion exchanges work? and what are examples of each
the positive ions stay bind to the negative charged exchanged
postively charge anion exchange - DEAE
negatively charged cation exchange- CM S columns
and the negative ions bind to the postive charged ions
when the protein sticks what are the two way to get it off
1.change pH using a gradient
2. changing the salt concentration
what is the ion exhange s coloums cation( +) elution order at PH 7
cytohrome c 12kd. 10pi
ribonuclease. 13.7 kd 7.8 pi
lysozyme 14.7 kd 9.6 pi
myoglobin 17 kd 7.5 pi
- nothing flows
- myo
- ribo
- lyo
5, cyto
A protein of intrest but unknown sequenxce does ntot bind to DEAE column at ph 8.5 which statements is true ?
A. the protein has many acidic residues
b. the pi of the protein is less than 8.5
c. the protein has more lys and arg residues than asp and glu residues ( perciprertation )
it is anion columns c bc lys and arg pi are greater that pH
what is used for the seperation based on affinity ?
atp columns are used to purify protein that bind to atp , these atp affinity protein bind the the column
what is another form to separate
polyacrylamide gel electrophoresis that seperates based on size that boils proteins in detergent sds and breaks down proteins and gives them a negative charge
what quantitative amino acid analysis ?
it is where we hydrolysize all amide bonds with 6N HCI and tells us the amino acids present but no sequence
what are the drawback of 6 HCL and what amino acids are missing from quantative amino acid analysis and why ?
Gln glutamine and Asn asparagine bc there have amide side chains the get conveted in to glutamic acid and aspartic acid,
what can also be used to hydrolyze amino acids ?
DABS , it provides us a floruscent derivitive
what is end group analysis
using reagents to indentify the AA at the N terminus or C and the number of polypetides in a chain
