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Why were cells resuspended in PBS before bing resuspended in SB?
- Rinse cells
- Maintains pH and prevents denaturation
What is sonification and how was it used?
Lyse cells without damaging proteins
Why centrifuge sonicated bacterial cells?
Remove insoluable cell debris
Why keep bacterial lysate cold?
- Prevent protease activity
- Preserve protein structure and function
Describe ion exchange chromatagraphy.
- Protein purification
- Beads with covalently bound ions to attract ionic proteins
Describe basis of DEAE sephadex chromatography
- DEAE sephadex is anion exchange (positive beads attracting anions - negative charged proteins)
- Positively charged molecules elute
- Elute anions with salt gradient or low pH
Describe gel filtration chromatography.
- Beads with pores of different sizes
- Large molecules elute first
After applying your sample to your DEAE column, you washed it checking the A280 every few fractions. Why?
- A280 is the simplest, quickest way to check for protein presence. CWB is used to wash out positive proteins.
- Wash until 280nm shows none left. Then move on to salt gradient to elute neg proteins
You next applied a series of buffers with increasing concentrations of NaCl. What is this step called? Why not increase NaCl to 1M in 1 step?
- "salting out"
- Doing it all at once would not lead to purification since they would all come out at the same time.
In class you performed spectrophotometric assays using jenway and microplate reader. Why is precise reproducibility more important with microplate reader?
Instrument measures absorbance top to bottom. Jenway shines across 1cm cuvette (already precise)
Doing DEAE column with 40 fractions, you start elution buffers after collecting fraction 15. Why?
Elution buffers start when A280 is back to almost zero, i.e. when unbound proteins have been completely eluted.
Explain differences between A280 and A595.
- A280 measures relative protein concentration and some DNA content.
- Bradford binds only to protein at A595.
Of A280 and A595, which is more reliable for protein concentration?
A595 because only protein concentration is measured (not DNA)
Why is A280 used to measure relative protein concentration fractions?
It's quick and easy
Which fractions contain cellulose digesting activity? Explain.
21-25. Product ethanol absorbs at 450nm
Which enzyme activity peak contains the majority of the cellulose digesting activity? Explain.
23 - highest 450nm peak
Breaks/denatures disulfide bonds
APS and TEMED
Initiates polymerization of acrylamide
- Denatures 2 and 3 structures and linearizes proteins.
- Provides uniform negative charge
Acrylamide an dbisacrylamide
- Provides matrix of gel.
- Determines "pore size" of gel.
What is(are) the MWs of the band(s) aossicated with cellulose digesting activity? Explain.
- 125kDa. All fractions except F17 show cellulose activity. This is shown on the graph where OD420 peaks are located.
- 50+75 = 125
You do gel filtration with G-100 column. ALL activity found in initial elution. Also see SDS-PAGE results. Explain both.
- Enzyme must have two parts connected with disulfide bonds. Total size is 125kDa, so it's eluted first in G100 column.
- Under reducing conditons in SDS-PAGE, it is shown in its two parts: 50 + 75. Under non-reducing, it's show in at 125kDa.