This protocol utilizes the coupling of Antibody to Protein
A Coated beads using DMP. This allows for elution of bound proteins and reuse
of beads, if desired.
Bio-Rad Protein A Support
Control IgG (depending on antibody source; rabbit, mouse,
PBSt (PBS + 0.1% Tween20)
0.2 M Sodium Borate pH 9.0
0.1M Glycine, pH 2.5
Trichloroacetic Acid (TCA)
1% Deoxycholic acid (DOC)
- Remove an appropriate amount of Protein A beads and
wash 3 times with PBSt. You will need a minimum of 2 tubes of beads at this
point, one for you’re your antibody of interest and one for control IgG.
- Assuming you are using 10-100 µl of beads, add PBSt
and antibody to a final volume of ~500µl.
- Mix the antibody/bead solution in the cold room for 1
hour to overnight.
- Wash the beads 3 times with PBSt
- Wash the beads 2 times with 0.2M Sodium Borate pH9.0
- With beads in Borate, prepare 20mM solution of
dimethylpimilidate (DMP) in sodium borate (you will need a minimum of 10
fold excess volume of this solution relative to your starting bead bed
volume, for example 50ul beads require a minimum of 500 µl of 20mM DMP
- Resuspend beads in at least a ten fold excess of
Borate + 20mM DMP. If I have less than 100µl of beads, I usually use
- Rotate at room temperature for 30 minutes.
- Spin down beads and wash 2-3 times in 100mM Tris pH8.
This step quenches the coupling reaction.
- Allow beads to rotate with tris for 2 hours at room
temperature, or overnight in the cold room.
- Wash beads 2-3 times in PBSt
- Wash beads 3 times in 0.1M glycine pH2.5. This step
removes any unbound antibody.
- Wash beads back into PBSt 3 times. The glycine will
effectively denature the antibodies bound to beads, so it is important to
let them refold in PBSt for a minimum of several hours to overnight in the
cold. If not the IP can be very dirty.
- Wash beads in buffer of choice prior to IP. Spin down
and remove excess buffer.
- Add solution you are IP-ing from to the beads,
resuspend and incubate at either room temperature or in cold for a minimum
of 1-2 hours. The length of time for the IP depends on the quality of the
antibody. Also, things will get degraded faster (much faster) at room
temperature. I would recommend starting with 2-3 hours in the cold.
- After the IP, wash the beads 3 times in buffer (PBSt
works, although you can wash with higher salt to be more stringent).
- Elute the beads 3 times in 0.1M Glycine pH 2.5.
Transfer the supernatant after each elution into 1.0M Tris pH8. Normally, I
will elute with 3 times, each with 200-300µl of glycine, and put all three
elutions into 300µl of 1M Tris (final volume of 1.2ml).
- TCA precipitate samples by adding a 1:50 volume of 1%
DOC and a 1:10 volume of TCA. Vortex quickly to mix.
- Store TCA precipitation on ice for 30 minutes to
overnight (30 minutes is almost always sufficient).
- Spin in a cold benchtop centrifuge at high speed for
- Remove supernatant, add 500µl of acetome to pellet to
remove salt and crap.
- Respin for 5-10 minutes on high speed.
- Remove all supernatant and resuspend in sample
buffer. If sample buffer doesn’t stay blue, that is bad.