Covalent conjugation The APC is covalently coupled to the IgG through reaction of the maleimide groups with the free sulfhydryl on the IgG.
For.5 ml sample volumes: NAP5 columns (Sephadex G-25 DNA grade code reduction location de voiture fr Amersham, catalog.
22360, mw 334.42 NEM N-Ethylmaleimide Sigma, Catalog E-1271, mw 125.1 dmso - anyhydrous dimethyl sulfoxide Aldrich, catalog.Hardy, RR: Purification and coupling of fluorescent proteins for use in flow cytometry.References and credits: This protocol is based on an original protocol devised by Randy Hardy, and modified by Alan Stall and Aaron Kantor.3624-01,.44 trizma.0 - Combination of Tris base and TrisHCl Sigma, catalog.Covalent conjugation The PE is covalently coupled to the IgG through reaction of the maleimide groups with the free sulfhydryl on the IgG.Preparation of APC, dialyze or exchange the APC into "Dialysis Buffer".The conjugation kit provides enough of all of the necessary materials to conjugate 1 mg of antibody; the entire procedure takes about cadeau original pour copine 2 hours to complete (the kit eliminates steps I and II in the conjugation protocol below).You should be familiar with how to use desalting columns and how to take absorbance spectra.Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer".Never freeze the congugates.
Note: These molar ratios (2 APC per IgG) have worked very well.
If you are only performing a limited number of PE conjugations, this alternative is probably desirable.
(1 mg/ml of APC has an OD at 655nm.9).
The hinge disulfide bonds are reduced to yield free sulfhydryls.
See the sample absorbance spectrum for APC.P1045 smcc - succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate Pierce, catalog.Note: PE is most stable as a SAS (sodium ammonium sulfate) precipitate prior to coupling.Direct PE conjugates are relatively easy to make.In: Handbook of Experimental Immunology, 4th.It emits at about 680 nm, and can be excited by either a dye laser (595-605 nm or the cheaper HeNe laser (633 nm).Pass the derivatized PE over a gel filtration column pre-equilibrated with "Exchange Buffer".Allophycocyanin can be purchased from several vendors, or isolated from the algae directly (be prepared to spend 2 weeks in the cold room).Typically, only one PE molecule is conjugated to an antibody.It may be useful to spin APC conjugates prior to use in staining, especially if background seems to be a problem (e.g., at 10,000g in a microcentrifuge, at 4C).NaHCO3 - sodium bicarbonate.
