TITLE: Antibody Purification and Labeling
AUTHORS: Maya Brewer, Yuantee Zhu, Danielle Gutierrez, Jeff Spraggins, Mark de Caestecker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the process for antibody purification and subsequent labeling for direct immunofluorescence.</div></div>

[STEPS]
?. [Antibody Purification]
For all preparations: this is to remove BSA, azide, or glycine that are often added by the manufacturer. If the antibody has nothing added, skip the purification step.
?. [Antibody Purification]
Assemble Amicon Pro Affinity Concentrator by carefully attaching the Amicon Ultra device to the exchange device.
?. [Antibody Purification]
Once the concentrator is assembled, remove the collection tube cap, lift the exchange device cap, and add 1 mL of 1X PBS to moisten the cellulose membrane of the Amicon Ultra device.
?. [Antibody Purification]
This wash will ensure that the antibody does not stick to the membrane upon its addition.
?. [Antibody Purification]
Centrifuge at 4000 x g for 3 minutes.
?. [Antibody Purification]
Add desired amount of antibody to the exchange device.
?. [Antibody Purification]
Centrifuge for 10 minutes at 4000 x g in a swinging-bucket rotor.
?. [Antibody Purification]
Add 1 mL of PBS to the device and centrifuge again at 4000 x g for 10 minutes twice, discarding the flow through each time.
?. [Antibody Purification]
Collect purified antibody from the device by reverse spin.
?. [Antibody Purification]
Place a collection tube on top of the Amicon Ultra-0.5 device.  Make sure flow through has been removed from bottom tube.
?. [Antibody Purification]
Invert the assembly and centrifuge in a fixed-angle rotor at 1000 x g for 2 minutes.
?. [Antibody Purification]
Use a nanodrop to measure the purified antibody at an absorbance of 280 nm.
?. [Antibody Purification]
Make sure that the sampling arm on the nanodrop is up.
?. [Antibody Purification]
Using a P10 pipette, add 1 µL of the sample onto the lower measurement pedestal.
?. [Antibody Purification]
Lower the sampling arm and measure the absorbance of the antibody at 280 nm (A280). The concentration should be given in mg/mL.
?. If labeling 100 ug or less of antibody, stop here and follow procedure from Antibody Labeling Kit.
?. If you have more than 100 ug of antibody, continue to the next section.
?. [Labeling Preparation- Dye]
Dissolve amine-reactive compound (NHS ester) in DMSO or DMF at 10 mg/mL (i.e. 100 µL of DMSO is added to 1 mg compound).
?. [Labeling Preparation- Dye]
Mix the solution by vortex.
?. [Labeling Preparation- Dye]
Aliquot solution into small Eppendorf tubes and store at -80C if all dye is not used during reaction.
?. [Antibody Labeling]
For successful labeling, protein concentration should definitely not be less than 2mg/ml, ideally 5-20 mg/mL.
?. [Antibody Labeling]
Antibodies should be in a buffer free of any amine-containing compounds (glycine, Tris, or ammonium ions) and stabilizing proteins (bovine serum albumin). These compounds will interfere with the labeling reaction.
?. [Antibody Labeling]
Add sodium bicarbonate to the antibody at a concentration of 0.1 M (i.e. 20 µL of sodium bicarbonate per 200 µg antibody) to make a slightly basic pH for the conjugation to occur.
?. [Antibody Labeling]
Add 1 µL of the chosen fluorophore NHS ester for each 200 µg of antibody.
?. [Antibody Labeling]
Labeling of antibody occurs by the Alexa Fluor dye linking to the primary amine (R-NH2) in peptides and proteins.
?. [Antibody Labeling]
Invert tube five times.
?. [Antibody Labeling]
Wrap the tube in foil and incubate at room temperature for 60 minutes on a shaker.
?. [Antibody Labeling]
Prepare the column for the purification of the labeled antibody during incubation step.
?. [Purifying the Labeled Antibody]
Assemble the spin column from the Antibody Conjugate Purification Kit.  
?. [Purifying the Labeled Antibody]
Remove the column lid.
?. [Purifying the Labeled Antibody]
Cut-off square tab at the bottom of the column.
?. [Purifying the Labeled Antibody]
Add both frits to the column and push them to the bottom of the column using a stir rod or P1000 pipette tip.
?. [Purifying the Labeled Antibody]
Use the yellow cap only when filling the column.
?. [Purifying the Labeled Antibody]
Stir the purification resin, then add 1 mL of the suspension into the column and allow it to settle by gravity.
?. [Purifying the Labeled Antibody]
Continue to add more of the suspension until the resin bed volume is about 1.5 mL.
?. [Purifying the Labeled Antibody]
Place the spin column in a collection tube, and place both in a 15 mL conical tube. Centrifuge at 1100 x g for 3 minutes.
?. [Purifying the Labeled Antibody]
Empty collection tube containing column buffer.
?. [Purifying the Labeled Antibody]
Add the sample to the center of the column, dropwise. Allow the solution to absorb into the resin bed.
?. [Purifying the Labeled Antibody]
Place the spin column into the empty collection tube and 15 mL conical tube.
?. [Purifying the Labeled Antibody]
Centrifuge at 1100 x g for 5 minutes.
?. [Purifying the Labeled Antibody]
Collect labeled antibody from the collection tube. Aliquot then store at 4˚C short term, and -20˚C long term.
?. [Purifying the Labeled Antibody]
The conjugates can survive freeze thawing but you will need to evaluate each new antibody you use to make sure. We always evaluate one freeze/thaw cycle by repeating staining.