imported>Virgilio |
imported>Xenbase |
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| Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]
| | ==Reporting a bug in Xenbase== |
| | If you find a bug in Xenbase, we would really like to know! |
| | However, it is very important that we can find your bug so that we can fix it. |
| | To help us find your bug, please fill out [http://xenbase.org/xenwiki/images/6/6e/Bug_report.txt this] form, and e-mail it to us! |
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|
| | | ==Bug Handling Process== |
| link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
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| IP Protocol
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| 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.
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| Materials:
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| Bio-Rad Protein A Support
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| Control IgG (depending on antibody source; rabbit, mouse, etc..)
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| PBSt (PBS + 0.1% Tween20)
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| 0.2 M Sodium Borate pH 9.0
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| 0.1M Glycine, pH 2.5
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| Trichloroacetic Acid (TCA)
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| 1% Deoxycholic acid (DOC)
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| Methods
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| 1. 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.
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| 2. Assuming you are using 10-100 µl of beads, add PBSt and antibody to a final volume of ~500µl.
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| 3. Mix the antibody/bead solution in the cold room for 1 hour to overnight.
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| 4. Wash the beads 3 times with PBSt
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| 5. Wash the beads 2 times with 0.2M Sodium Borate pH9.0
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| 6. 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 solution).
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| 7. Resuspend beads in at least a ten fold excess of Borate + 20mM DMP. If I have less than 100µl of beads, I usually use 500-1000µl regardless.
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| 8. Rotate at room temperature for 30 minutes.
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| 9. Spin down beads and wash 2-3 times in 100mM Tris pH8. This step quenches the coupling reaction.
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| 10. Allow beads to rotate with tris for 2 hours at room temperature, or overnight in the cold room.
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| 11. Wash beads 2-3 times in PBSt
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| 12. Wash beads 3 times in 0.1M glycine pH2.5. This step removes any unbound antibody.
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| 13. 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.
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| 14. Wash beads in buffer of choice prior to IP. Spin down and remove excess buffer.
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| 15. 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.
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| 16. After the IP, wash the beads 3 times in buffer (PBSt works, although you can wash with higher salt to be more stringent).
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| 17. 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).
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| 18. TCA precipitate samples by adding a 1:50 volume of 1% DOC and a 1:10 volume of TCA. Vortex quickly to mix.
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| 19. Store TCA precipitation on ice for 30 minutes to overnight (30 minutes is almost always sufficient).
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| 20. Spin in a cold benchtop centrifuge at high speed for 15 minutes.
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| 21. Remove supernatant, add 500µl of acetome to pellet to remove salt and crap.
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| 22. Respin for 5-10 minutes on high speed.
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| 23. Remove all supernatant and resuspend in sample buffer. If sample buffer doesn’t stay blue, that is bad.
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