1.0 1234361747009 OMERO.editor PROTOCOL_ARCHIVE Fluorophore Conjugation of Antibodies (Lamond Lab) This protocol has been adapted from the Lamond Lab protocol found here: http://www.lamondlab.com/f7protocols.htm Materials: In addition to the [[0]] you wish to label and the [[1]] (described below) you will need: Antibody name 0 OPTIONAL TEXT Fluorophore 1 OPTIONAL ENUMERATION Alexa Fluor 488 Alexa Fluor 488 Alexa Fluor 546 STEP_GROUP Fluorophores: Alexa Fluor 488 / Alexa Fluor 546 (succinimidyl esters), Molecular Probes, Cat.No. A-20000 / A-20002, dissolved in anhydrous DMSO; SINGLE_STEP Phosphate-buffered saline (PBS); Sodium bicarbonate, 1M; SINGLE_STEP PD-10 column, Sephadex G-25M, AmershamPharmacia, Cat.No. 17-0851-01; Bio-Spin 6 columns, BioRad, Cat.No. 732-6002 SINGLE_STEP Protein Preparation: Antibodies in crude serum will not label well. IgG can be affinity purified by using Protein A or Protein G. Purified IgG should be in a buffer free of ammoum ions or primary amines. Replace unsuitable buffer (e.g. Tris or glycine) with PBS by dialysis or another method. The presence of low concentrations (less than 3mM) of sodium azide will not interfere with the conjugation reaction. SINGLE_STEP Labeling Reaction: To [[2]] of a 2 mg/ml IgG solution (in 2 ml Eppendorf tube), add [[3]] of 1M sodium bicarbonate (bicarbonate is added to raise the pH of the reaction mixture, since succinimidyl esters react efficiently at pH 7.5-8.5). Add [[4]] of reactive Alexa dye (see test reactions), mix the reaction by inverting the tube a few times and incubate the reaction mixture on a rotating wheel for 1 hour at room temperature followed by an overnight incubation at 4 °C. IgG volume 2 OPTIONAL NUMERIC 2 2 ml Sodium Bicarbonate vol 3 OPTIONAL NUMERIC 100 100 µl Alexa dye vol 4 OPTIONAL NUMERIC µl STEP_GROUP Test Reactions When first conjugating an antibody to Alexa Fluor 488 or Alexa Fluor 546, a range of Alexa dye to antibody concentrations should be compared. For a first-time titration of Alexa dye we recommend a range of 10 to 200 µg Alexa dye per mg antibody. Compare each conjugate by cell staining.Select the conjugate with the brightest “positive” cells which still has low background on “negative” cells. SINGLE_STEP Purification of the conjugated Antibody: Non-conjugated Alexa dye can be removed by gel filtration using a PD-10 column (Sephadex G-25M). Elute the conjugated antibody with PBS (optionally with 0.01% sodium azide). For the test reactions Bio-Spin 6 columns can be used. SINGLE_STEP