Protein purification and analysis

Question 1

protein structure dimensions

Answer 1

1- linear amino acid sequence of polypeptide
2- local structure of linear segments of polypeptide backbone atoms
3- 3D arrangement of all atoms
4- arrangement of separate polypeptide chains into functional protein

Question 2

Protein purification (3)

Answer 2

Size Exclusion Chromatography (molecular weight): large molecules can´t enter pores and are the first to pass column

Ion exchange chromatography (pI): beads have a charge and bind to opposite (anion exchange binds -, cation exchange binds +) that will take more time to flow

Column chromatography- matrix separate fractions of molecules

combine methods to obtain pure protein, important to sequence aa first

Question 3

Affinity Chromatography and tags

Answer 3

when using recombinant protein expression, vectors have affinity tag, 2nd method needed

1- Hexa histidine binds to Ni2+ and when elutes with imidazole protein attached to histidine will be released
2- GST (25kDa - problem disruption) binds to gluthatione, elutes with free reduced gluthatione
3-FLAG (small peptide) binds to antibody FLAg and elution with free flag

Question 4

Detection of proteins (4 direct and 3 indirect)

Answer 4

Direct
1- absorbance: most at 280nm (not good to discriminate). Bradford assay - blue Amax=595 (comassie)
2- SDS-page: separates by mass
3- Western Blot: Ab specific for protein of interest or affinity tag
4- MS

Indirect: enzymatic activity, detecting metalloproteins, DNA/RNA binding assays

Question 5

Applications of pure proteins (4)

Answer 5

1- structural biology: NMR, X-ray, cryo-EM
2- binding assays (interaction between proteins): GST pulldown or biophysical methods
3-enzymatic assays
4- functional assays (phosphorylation)