User:Reshma P. Shetty/Scratchpad

Random notes to keep track of

Culture conditions

• 90μM ZnCl₂ (Barbas lab papers) or 100 μM 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 MJ, Nierman WC, Wiggs J, and Neff N. A quantitative assay for bacterial RNA polymerases. J Biol Chem. 1979 Oct 25;254(20):10061-9.
2. Eron L and Block R. Mechanism of initiation and repression of in vitro transcription of the lac operon of Escherichia coli. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1828-32. DOI:10.1073/pnas.68.8.1828 |
3. Uptain SM. Assay of intrinsic transcript termination by E. coli RNA polymerase on single-stranded and double-stranded DNA templates. Methods Enzymol. 2003;371:339-51. DOI:10.1016/S0076-6879(03)71025-8 |
4. Vo NV, Hsu LM, Kane CM, and Chamberlin MJ. In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape. Biochemistry. 2003 Apr 8;42(13):3798-811. DOI:10.1021/bi026962v |
5. Wang QP and Kaguni JM. Transcriptional repression of the dnaA gene of Escherichia coli by dnaA protein. Mol Gen Genet. 1987 Oct;209(3):518-25. DOI:10.1007/BF00331158 |

Chromosomal integration

6. Haldimann A and Wanner BL. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J Bacteriol. 2001 Nov;183(21):6384-93. DOI:10.1128/JB.183.21.6384-6393.2001 |
7. Platt R, Drescher C, Park SK, and Phillips GJ. Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome. Plasmid. 2000 Jan;43(1):12-23. DOI:10.1006/plas.1999.1433 |
8. Katz L, Brown DP, and Donadio S. Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea. Mol Gen Genet. 1991 May;227(1):155-9. DOI:10.1007/BF00260721 |
9. Hasan N, Koob M, and Szybalski W. Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori- plasmids and their in vivo chromosomal integration and retrieval. Gene. 1994 Dec 2;150(1):51-6. DOI:10.1016/0378-1119(94)90856-7 |
10. Diederich L, Rasmussen LJ, and Messer W. New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome. Plasmid. 1992 Jul;28(1):14-24. DOI:10.1016/0147-619x(92)90032-6 |
11. Le Borgne S, Bolívar F, and Gosset G. Plasmid vectors for marker-free chromosomal insertion of genetic material in Escherichia coli. Methods Mol Biol. 2004;267:135-43. DOI:10.1385/1-59259-774-2:135 |

Protein purification

Notes from the following book ...

12. ISBN:0387940723

Chromatography

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

Concentration

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[13]

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

Buffer exchange

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

References

13. Saul A and Don M. A rapid method of concentrating proteins in small volumes with high recovery using Sephadex G-25. Anal Biochem. 1984 May 1;138(2):451-3. DOI:10.1016/0003-2697(84)90838-8 |
14. Helmerhorst E and Stokes GB. Microcentrifuge desalting: a rapid, quantitative method for desalting small amounts of protein. Anal Biochem. 1980 May 1;104(1):130-5. DOI:10.1016/0003-2697(80)90287-0 |

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#Notes

Native purifications

Knight:Purification_of_His-tagged_proteins/Native#Notes

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)
   • The protein should refold on the column.
4. 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

• Bioinstrumentation facility

Some samples to compare

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)

Other affinity tags

IMPACT from NEB

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

MBP from NEB

• pMAL Protein Fusion and Purification System

Zinc proteins

• Generally useful info

Gel shifts

Segal et al. 2006

20 mL of binding reactions <-- must be a typo likely 20μL

• 10 mM Tris (pH 7.5)
• 90 mM KCl
• 1 mM MgCl₂
• 100 μM ZnCl₂
• 1% (w/v) BSA
• 5 mM DTT
• 0.12 μg/μL 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

15. Segal DJ, Crotty JW, Bhakta MS, Barbas CF 3rd, and Horton NC. Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. J Mol Biol. 2006 Oct 20;363(2):405-21. DOI:10.1016/j.jmb.2006.08.016 |

Segal et al. 1999

• ZBA
  • 10 mM Tris pH7.5
  • 90 mM KCl
  • 90 μM ZnCl₂
  • 1 mM MgCl₂
• 1% BSA
• 5 mM DTT
• 0.12 μg/μL 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

16. Segal DJ, Dreier B, Beerli RR, and Barbas CF 3rd. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2758-63. DOI:10.1073/pnas.96.6.2758 |

Protein purification

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 μM ZnCl₂
• 1 mM MgCl₂

16. Segal DJ, Dreier B, Beerli RR, and Barbas CF 3rd. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2758-63. DOI:10.1073/pnas.96.6.2758 |

Crotty et al. 2005

Resuspend cell pellet in cold ZBA

1. Inoculate 200 ml culture grown overnight in LB media containing 50 μg/mL ampicillin and 90 μM ZnCl₂ into 1 L LB containing 50 μg/ml ampicillin and 90 μM 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 μM ZnCl₂, 1mM MgCl₂, 90mM KCl) and sonicated at 277 K with 50% output for 20 1 s pulses.
8. Remove 20 μl 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 μm filter followed by an additional filtration using a 0.2 μm 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
    • 20 mM Tris–HCl pH 8.0
    • 20 mM NaCl
    • 2 mM CaCl₂
    • 90 μM ZnCl₂
19. Add factor Xa protease at a concentration of 0.001 mg per mL of fraction volume
20. Agitate overnight at room temperature.
21. Verify cleavage of the MBP tag from Aart by SDS–PAGE
22. Dilute the cleavage reaction 1:4 in ZBA
23. Load onto a heparin column pre-equilibrated in ZBA
24. Elute protein with a 2 M KCl gradient
25. Identify fractions containing protein by SDS–PAGE
26. Passage over a MonoQ column (Amersham Inc.) equilibrated in ZBA to remove co-purifying DNA

Reference

17. Crotty JW, Etzkorn C, Barbas CF 3rd, Segal DJ, and Horton NC. Crystallization and preliminary X-ray crystallographic analysis of Aart, a designed six-finger zinc-finger peptide, bound to DNA. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Jun 1;61(Pt 6):573-6. DOI:10.1107/S1744309105014363 |
18. Segal DJ, Crotty JW, Bhakta MS, Barbas CF 3rd, and Horton NC. Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. J Mol Biol. 2006 Oct 20;363(2):405-21. DOI:10.1016/j.jmb.2006.08.016 |

β-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.
   • 96 well assay of β-galactosidase activity in cells.
   • Monitors rate of production of ONPG rather than doing an end-point assay.
2. In vitro kinetics of β-galactosidase by Ursell et al.
   • Uses purified protein
3. Kinetic Analysis of β-Galactosidase Activity
4. Beer's law
5. Promega β-galactosidase assays - 96 well format and standard curves
6. Beta-galactosidase control enzyme
7. β-galactosidase assay - 96 well format
8. Thesis on β-galactosidase from Lactobacillus plantarum

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

Pulling together data from multiple plate reader OD runs:

cat 20071108-RS\ -\ OD* | awk '/\tM\t/' | sort > ODdata.txt

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.