By Amanda E.I. Proudfoot, Timothy N.C. Wells, Christine Power
Specialist investigators describe intimately the main everyday ideas in chemokine biology. protecting either ligands and receptors, those effectively reproducible equipment disguise all points of chemokine study, starting from the cloning and characterization of chemokines and their receptors, by using animal types to review chemokine functionality in vivo. each one process additionally contains correct heritage info, in addition to supplying an invaluable bibliography that renders the examine of chemokines available in any respect degrees of expertise. finished and hugely functional, Chemokine Protocols bargains experimental and scientific chemokine researchers modern-day gold-standard choice of confirmed tools for reading this biologically ubiquitous and significant classification of proteins.
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Additional info for Chemokine Protocols
Cloning of the Chemokine Gene into pET24d We routinely clone chemokines using polymerase chain reaction (PCR). The small size of chemokine genes, the frequent addition of N-terminal or C-terminal tags, and the need to localize precisely the initiator methionine codon in bacteria expression systems are all most easily dealt with by amplifying the genes. In this section, it is assumed that the reader has a grasp of basic cloning techniques (18) and is familiar with simple PCR amplification (21). 1.
3. Incubate at room temperature for 1 h on a rocking platform. 4. Remove the inoculate and rinse with 5 mL of Ex-Cell 400 medium. 5. Add 30 mL of Ex-Cell 400 medium and incubate at 27°C for 48 h. 6. 22-µm filter, and store at 4°C for purification. 4. Notes 1. Insect cells will grow reasonably well at any temperature from room temperature to 30°C and they do not require CO2. An optimal growth temperature is 27°C. In order to maintain consistency of cell growth and virus infection, cells should be used within 20–30 passages.
3. 4. 5. 6. 7. 8. 9. Hydrogen fluoride 4 L tank (Matheson Gases Canada, Edmonton AB). Cresol, thiocresol, dimethyl sulfide, Piperidine (Aldrich, Milwaukee, MI). HF apparatus with all Teflon or Kel-F construction reaction vessels. Calcium oxide trap (Peptides International, Louisville, KY). Vacuum pump and KOH trap. All Teflon 47mm filter holder (Savillex, Minnetonka, MN). Zitex filter sheets no A115 (Chemplast, Wayne, NJ). Shoulder length and standard neoprene gloves, and neoprene apron. Face shield (Nalgene, Rochester NY).