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

[DESCRIPTION]
This protocol describes the process for antibody purification, subsequent labeling for direct immunofluorescence, and validating antibody labeling.

[STEPS]
SECTION: Antibody Purification
2. Prepare buffers from AB buffer kit:
SECTION: Antibody Purification
1. This is to remove any BSA, azide, glycine that is often added by the
manufacturer for stabilization. If the antibody has nothing added, skip this section.
SECTION: Antibody Purification
2.1. 10X binding buffer must be diluted to 1X (9 parts ddH 2 O and 1 part 10X binding buffer).
SECTION: Antibody Purification
2.2. 10X elution buffer must be diluted to 1X (9 parts ddH 2 O and 1 part 10X elution buffer).
SECTION: Antibody Purification
2.3. Neutralizing buffer does not have to be diluted in any way. Prepare two collection tubes containing 30 µl of neutralizing buffer for the eluted fractions.
SECTION: Antibody Purification
3. Resuspend medium of spintrap column
SECTION: Antibody Purification
4. Remove the bottom cap from the column and save for later use.
SECTION: Antibody Purification
5. Place the column in a 2 mL collection tube and centrifuge at 100 x g for 30 seconds to remove storage solution.
SECTION: Antibody Purification
6. Add 600 ul of binding buffer to the tube
SECTION: Antibody Purification
7. Centrifuge at 100 x g for 30 seconds
SECTION: Antibody Purification
8. Add no more than 600 uL of the anitbody to the tube
SECTION: Antibody Purification
8.1. Several sample applications can be made subsequently as long as the capacity of the column is not exceeded.
SECTION: Antibody Purification
9. Secure the top and bottom cap on top the tube and incubate for 4 minutes while gently mixing
SECTION: Antibody Purification
10. Remove the bottom cap and centrifuge the column at 100 x g for 30 seconds
SECTION: Antibody Purification
11. Add 600 ul of binding buffer to the column to wash
SECTION: Antibody Purification
12. centrifuge at 100 x g for 30 seconds
SECTION: Antibody Purification
13. Add 600 ul of the binding buffer to the column for a second wash
SECTION: Antibody Purification
14. Centrifuge at 100 x g for 30 seconds
SECTION: Antibody Purification
15. Add 400 ul of elution buffer and mix by inverting tube several times
SECTION: Antibody Purification
16. Place the column in one of the 2 ml collection tubes containing 30 ul of neutralizing buffer
SECTION: Antibody Purification
17. Centrifuge at 100 x g for 30 seconds
SECTION: Antibody Purification
18. Add 400 ul of elution buffer and mix by inverting tube several times
SECTION: Antibody Purification
20. Centrifuge at 100 x g for 30 seconds
SECTION: Antibody Purification
19. Place the column in the other 2 ml collection tube containing 30 ul of neutralizing buffer
SECTION: Buffer Exchange
21. Assemble Amicon Pro Affinity Concentrator by carefully attaching the Amicon Ultra device to the exchange device
 
 

SECTION: Buffer Exchange
22. Once the concentrator is assembled, remove the collection tube cap, life the exchange device cap, and add 1 ml of binding buffer or PBS to moisten the cellulose membrane of the Amicon Ultra device. This wash ensures that the antibody does not stick to the membrane upon its addition.
SECTION: Buffer Exchange
23. Centrifuge at 4000 x g for 3 minutes
SECTION: Buffer Exchange
24. Add 1 ml binding buffer or PBS and antibody collected in neutralizing buffer to the exchange device
SECTION: Buffer Exchange
25. Centrifuge for 15 minutes at 4000 x g in a swinging-bucket rotor
SECTION: Buffer Exchange
26. Add 2 mL of binding buffer or PBS to the device and centrifuge again at 4000 x g for 10 minutes twice
SECTION: Buffer Exchange
27. Collect purified antibody from the device by reverse spin
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27.1. Place a collection tube on top of the Amicon Ultra-0.5 device
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27.2. Invert the assembly and centrifuge in a fixed-angle rotor at 1000 x g for 2 minutes
SECTION: Buffer Exchange
28. Use a nanodrop to measure the purified antibody at an absorbance of 280 nm
SECTION: Buffer Exchange
28.1. Make sure that the sampling arm on the nanodrop is up
SECTION: Buffer Exchange
28.2. Using a P10 pipette, add 2 µL of the sample onto the lower measurement pedestal
SECTION: Buffer Exchange
28.3. Lower the sampling arm and measure the absorbance of the antibody at A280. The
concentration should be given in mg/mL
SECTION: Labeling Protocol
29. 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. Labeling of antibody occurs by the DyLight dye linking to the primary amine (R-NH 2 ) in peptides and proteins.
SECTION: Labeling Protocol
30. Follow labeling kit protocol:
https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0011587_DyLight_Microscale_Antibody_Label_UG.pdf
SECTION: Labeling Protocol
31. Collect labeled antibody from the collection tube. Aliquot then store at 4˚C short term, and -20˚C long term.
SECTION: Labeling Protocol
31.1. *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
SECTION: Validating Antibody Labeling
32. After labeling antibodies, we initially validate antibody labeling by staining human kidney sections with the fluorophore conjugated antibody at different dilutions, as described in the antibody validation protocol (dx.doi.org/10.17504/protocols.io.5jyl8pwwdg2w/v1). We also compare staining by indirect immunofluorescence on the same section by indirect immunofluorescence using both the conjugated and original unconjugated antibody.
SECTION: Validating Antibody Labeling
33. If we show that the conjugated and unconjugated antibody is staining the tissue using indirect immunofluorescence but the directly conjugated fluorophore is undetectable, this suggests that the signal from the fluorophore is too low (i.e.: there may be too little dye conjugation). Conversely, if antibody staining is detectable with unconjugated antibodies, but the conjugated antibody is undetectable using both methods, it is possible too much dye is conjugated to the antibody and is inhibiting the epitope binding sites. To address this, we evaluate the dye: antibody ratio and repeat the conjugation reaction with different ratios of dye to antibody in the conjugation reaction depending on the results, as described below
SECTION: Validating Antibody Labeling
33.1. The dye to antibody ratio measures the average number of dye molecules conjugated to each antibody molecule using serial dilutions of the conjugated antibody in PBS, and measuring the absorbance of the conjugate at A280 and the maximum excitation wavelength for the dye.
SECTION: Validating Antibody Labeling
34. If the antibody was conjugated in house, and we show that the conjugated and unconjugated antibody is staining the tissue using indirect immunofluorescence but the directly conjugated fluorophore is undetectable, this suggests that the signal from the fluorophore is too low (i.e.: there may be too little dye conjugation). Conversely, if antibody staining is detectable with unconjugated antibodies, but the conjugated antibody is undetectable using both methods, it is possible too much dye is conjugated to the antibody and is inhibiting the epitope binding sites. 
SECTION: Validating Antibody Labeling
34.1. If the antibody was conjugated commercially, and we show that the unconjugated stains the tissue while the conjugated antibody does not it is possible there is either too little or too much dye conjugated to the antibody. If this occurs consider ordering a different antibody, a different lot of the same fluorophore conjugated antibody, or a different fluorophore conjugate, and retest
SECTION: Validating Antibody Labeling
33.2. Ideally, the optimal dye to antibody ratio is around 2, meaning there are about 2 molecules of dye per molecule of antibody. Some dyes such as the Alexa Fluors allow higher ratios upwards to 5-8 without overlabeling the antibody. On this basis, if the dye to antibody ratio is >2 and staining does not work, we repeat antibody conjugation reaction with reduced dye: antibody ratios. If the ratio of dye to antibody is > 2, then labeling is repeated after addressing the successful purification of the antibody, followed by using different ratio of dye to antibody, or decreasing the incubation time of the labeling reaction
SECTION: Validating Antibody Labeling
33.3. Section D of this document outlines instructions for calculating the dye to antibody ratio
https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0011587_DyLight_Microscale_Antibody_Label_UG.pdf