User:Reshma P. Shetty/Scratchpad

Random notes to keep track of

Culture conditions

 * 90&mu;M ZnCl2 (Barbas lab papers) or 100 &mu;M ZnCl2 (Pabo lab papers)

In vitro transcription

 * E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre protocol
 * RNA Polymerase from Escherichia coli from Sigma Aldrich


 * 1) Chamberlin-JBC-1979 pmid=114520
 * 2) Eron-PNAS-1971 pmid=4331560
 * 3) Uptain-MethodsEnzymol-2003 pmid=14712712
 * 4) Vo-Biochem-2003 pmid=12667071
 * 5) Wang-MolGenGenet-1987 pmid=2828882

Chromosomal integration

 * 1) Haldimann-JBacteriol-2001 pmid=11591683
 * 2) Platt-Plasmid-2000 pmid=10610816
 * 3) Katz-MolGenGenet-1991 pmid=2046656
 * 4) Hasan-Gene-1994 pmid=7959062
 * 5) Diederich-Plasmid-1992 pmid=1387714
 * 6) LeBorgne-MethodsMolBiol-1994 pmid=15269421

Protein purification
Notes from the following book ...
 * 1) Scopes isbn=0387940723

Chromatography

 * even packing and constant, even flow through the column is key to good results

Precipitation

 * ammonium sulfate or sometimes acetone is used to precipitate protein and resuspend in a smaller volume
 * must remove precipitant
 * precipitation usually only works for protein solutions with concentration > 1mg/mL.
 * lower concentrations eithr don't precipitate or denature

Adsorption to an ion exchanger

 * good for dilute solutions

Dialysis

 * semi-permeable membrane removes water
 * ultrafiltration
 * very fast and effective on dilute solutions
 * centrifugal filtration
 * low volumes

Gel filtration

 * at least 10-15% loss of protein can be expected
 * can use centrifugation for small volumes Saul-AnalBiochem-1984

Osmotic removal

 * sample is placed in dialysis tubing and immersed in solution or powder like PEG
 * be careful not to contaminate protein solution with polymer solution

Dialysis

 * often need multiple dialysis steps
 * proteolytic degradation may occur
 * slow

Gel filtration

 * preequilibrate column with buffer that the protein is to be exchanged into
 * rapid "desalting" occurs in one pass if the sample volume if < 1/5 column volume
 * small columns should be packed with finer grade beads to retain optimal resolution
 * rapid desalting of small samples with no dilution can be achieved by centrifugal method. Column of Sephadex G-25 is used inside a benchtop centrifuge.
 * sample should not have too much salt or protein for optimum separation
 * protein concentration should be < 30 mg/mL
 * doesn't matter what the column is washed with since protein travels before solvent front
 * final volume may be larger than initial volume before desalting
 * for buffer exchange, if protein concentration is low and volume large, "salt out" first and dissolve in a small volume of buffer and then use a relatively small desalting column

Notes from chat with Kathleen
Thanks Kathleen! Note that any errors below are mine.

Check solubility of protein
Talk:Knight:Protein solubility

General issues with protein purifications
Knight:Purification_of_His-tagged_proteins

Native purifications
Knight:Purification_of_His-tagged_proteins/Native

Refolding a protein on the column

 * 1) Lyse cells in denaturing buffer supplemented with 10 mM imidazole
 * 2) Run through column
 * 3) Wash with "native" wash buffer (contains 20mM imidazole)
 * 4) *The protein should refold on the column.
 * 5) Elute in "native" elution solution.

Centrifugal filtrations

 * Even though they are not supposed to, sometimes these columns can end up concentrating your buffers. This may explain why my dye front runs oddly when loading samples from the microcon.  The salt content has been increased.
 * These columns tend to bind an absolute amount of protein, not a fraction of what you send through it. Hence, larger scale preps tend to be preferable.

Circular dichroism
Some samples to compare
 * Bioinstrumentation facility
 * 1) Protein purified via denaturing prep with gel filtration buffer exchange into protein DNA binding buffer without BSA
 * 2) Negative control purified via denaturing prep with gel filtration buffer exchange into protein DNA binding buffer without BSA
 * 3) Protein purified via refolding prep with gel filtration buffer exchange into protein DNA binding buffer without BSA
 * 4) Negative control purified via refolding prep with gel filtration buffer exchange into protein DNA binding buffer without BSA
 * 5) protein DNA binding buffer without BSA
 * 6) Samples +/- DNA
 * 7) Samples +/- Zinc (or +/- EDTA)

IMPACT from NEB

 * IMPACT-CN System
 * Manual
 * Technical Bulletin
 * FAQ

MBP from NEB

 * pMAL Protein Fusion and Purification System

Zinc proteins

 * Generally useful info

Segal et al. 2006
20 mL of binding reactions <-- must be a typo likely 20&mu;L
 * 10 mM Tris (pH 7.5)
 * 90 mM KCl
 * 1 mM MgCl2
 * 100 &mu;M ZnCl2
 * 1% (w/v) BSA
 * 5 mM DTT
 * 0.12 &mu;g/&mu;L sheared herring sperm DNA (Sigma)
 * 10% (v/v) glycerol
 * 1 pM target oligonucleotide


 * Used 11 three-fold serial dilutions of protein.
 * Incubated 3h at room temperature
 * Resolved on a 5% polyacrylamide gel in 0.5x TBE buffer


 * 1) Segal-JMB-2006 pmid=16963084

Segal et al. 1999

 * ZBA
 * 10 mM Tris pH7.5
 * 90 mM KCl
 * 90 &mu;M ZnCl2
 * 1 mM MgCl2
 * 1% BSA
 * 5 mM DTT
 * 0.12 &mu;g/&mu;L sheared herring sperm DNA
 * 10% glycerol
 * 1 pM target oligonucleotide


 * Used 11 three-fold serial dilutions of protein.
 * Incubated 3h at room temperature
 * Resolved on a 5% polyacrylamide gel in 0.5x TBE buffer


 * 1) Segal-PNA-1999 pmid=10077584

Segal et al. 1999

 * Use ZBA/5 mM DTT was used as the column buffer with Protein Fusion and Purification System (New England Biolabs)

Zinc buffer A (ZBA)
 * 10 mM Tris pH7.5
 * 90 mM KCl
 * 90 &mu;M ZnCl2
 * 1 mM MgCl2


 * 1) Segal-PNA-1999 pmid=10077584

Crotty et al. 2005
Resuspend cell pellet in cold ZBA


 * 1) Inoculate 200 ml culture grown overnight in LB media containing 50 &mu;g/mL ampicillin and 90 &mu;M ZnCl2 into 1 L LB containing 50 &mu;g/ml ampicillin and 90 &mu;M ZnCl2.
 * 2) Grow cultures at 310 K until OD600nm 0.7
 * 3) Induce expression by adding 0.3 mM IPTG (final concentration).
 * 4) Grow cultures for an additional 21 h at 310 K
 * 5) Harvest by centrifugation at 5200 rev min 1 on a Sorvall HG-4L rotor for 25 min.
 * 6) Store cell pellets at 193 K until needed.
 * 7) Resuspend cell pellet from 4 L growth culture in an adequate volume (3 ml per gram of wet cells; 60 ml in this case) of cold ZBA (20 mM Tris pH 7.5, 90 &mu;M ZnCl2, 1mM MgCl2, 90mM KCl) and sonicated at 277 K with 50% output for 20 1 s pulses.
 * 8) Remove 20 &mu;l sample, spin to remove cell debris and measure absorbance at 280 nm.
 * 9) Continue Sonication in this way until the A280nm remained constant.
 * 10) A total of 20 sonication sessions was necessary to completely disrupt the cells.
 * 11) Centrifuge cell lysate for 60 min at 12 500g
 * 12) Filter using a 0.8 &mu;m filter followed by an additional filtration using a 0.2 &mu;m filter.
 * 13) Dilute filtrate 1:1 with ZBA.
 * 14) Load onto a 115 ml amylose resin (New England Biolabs) column.
 * 15) Wash the column with ten column volumes of ZBA.
 * 16) Elute protein with 50 ml 10 mM maltose.
 * 17) Pool fractions and concentrate in a YM-10 Centricon (Amicon Inc.).
 * 18) Dilute concentrated fraction pool with cleavage buffer
 * 19) *20 mM Tris–HCl pH 8.0
 * 20) *20 mM NaCl
 * 21) *2 mM CaCl2
 * 22) *90 &mu;M ZnCl2
 * 23) Add factor Xa protease at a concentration of 0.001 mg per mL of fraction volume
 * 24) Agitate overnight at room temperature.
 * 25) Verify cleavage of the MBP tag from Aart by SDS–PAGE
 * 26) Dilute the cleavage reaction 1:4 in ZBA
 * 27) Load onto a heparin column pre-equilibrated in ZBA
 * 28) Elute protein with a 2 M KCl gradient
 * 29) Identify fractions containing protein by SDS–PAGE
 * 30) Passage over a MonoQ column (Amersham Inc.) equilibrated in ZBA to remove co-purifying DNA

Reference

 * 1) Crotty-ActaCrystallographSectFStructBiolCrystCommun-2005 pmid=16511099
 * 2) Crotty-JMolBiol-2006 pmid=16963084

&beta;-galactosidase assays
See Beta-galactosidase and Beta-galactosidase assay


 * 1) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly by Wright et al.
 * 2) *96 well assay of &beta;-galactosidase activity in cells.
 * 3) *Monitors rate of production of ONPG rather than doing an end-point assay.
 * 4) In vitro kinetics of &beta;-galactosidase by Ursell et al.
 * 5) *Uses purified protein
 * 6) Kinetic Analysis of &beta;-Galactosidase Activity
 * 7) Beer's law
 * 8) Promega &beta;-galactosidase assays - 96 well format and standard curves
 * 9) Beta-galactosidase control enzyme
 * 10) &beta;-galactosidase assay - 96 well format
 * 11) Thesis on &beta;-galactosidase from Lactobacillus plantarum

To make IPTG/Xgal plates after the plates have already been poured, plate 40&mu;L 20mg/mL X-gal and 80&mu;L 50mM IPTG. Incubate at least an hour at 37&deg;C to let diffuse.

Pulling together data from multiple plate reader OD runs:

Miscellaneous
Accessing journals off-campus: Prefix url with http://libproxy.mit.edu/login?url=

Dilute 2-log ladder 100-fold in TE 10:1 for egels.