Sepharose Anti-GFP 抗体 (ab69314)

Due to the very high titer, assume near stochiometric binding: 1 nmole antibody will bind 2 nmole GFP. For example, 150ug antibody (since M.W. 150kDa) will bind 50ug GFP (since GFP M.W. ˜25kDa and IgG have two Fabs).

We typically recommend using 20ul of slurry per IP. Taking into account this volume as well as the number of IPs you expect to conduct at any one time should give you a good idea of what volumes to make your aliquots.

The exact protocol will vary and depends on the expression level of the GFP fusion and the type of protein and the purpose of the experiment.

The full protocol can be found in Megan C. Yap*#, Morris A. Kostiuk*, Dale D. O. Martin, Maneka A. Perinpanayagam, Pieter G. Hak, Anjaiah Siddam, Janaki R. Majjigapu, Gurram Rajaiah, Bernd O. Keller, Jennifer A. Prescher, Peng Wu, Carolyn R. Bertozzi, John R. Falck and Luc G. Berthiaume (2010) “Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry” J. Lipid Res. 51:1566-80.

Adapted from Yap et al. above...
Immunoprecipitation
Cells ( 1 °— 10 6 to 1 °— 10 7 ) were washed with cold PBS, harvested, and lysed with cold EDTA-free RIPA buffer by rocking for 15 min at 4°C. Cell lysates were centrifuged at 16,000 g for 10 min at 4°C and the postnuclear supernatants were collected.
[0.1% SDS, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium-
deoxycholate, 2 mM MgCl 2 , EDTA-free complete protease inhibitor]
GFP fusion proteins were immunoprecipitated from approximately 1 mg of protein lysates (depending on the expression level of the GFP fusion larger amount of lysate may be needed) with affinity purifi ed goat anti-GFP cross-linked to Sepharose beads by rocking for 2 h or overnight at 4°C. ...the rabbit anti-GFP-sepharose sold by Abcam can also be used.
The beads were extensively washed with 0.1% SDS-RIPA, resuspended
in 50 mM HEPES, pH 7.4, with 1% SDS containing buffer or any standard SDS-PAGE loading buffer depepnding on the application or purpuse of the experiment, and heated for 15 min at 80°C (or 5 min at 95C or 2min at 100C (again depending on the application).
. The supernatants containing the fusion proteins were collected.
Please note that this would be a guideline only and may require some individual optiization.

ab69314 is 100% compatible with the detergents that you mentioned.

To our knowledge, this antibody has not been specifically tested for co-IP of plant proteins. It will, however, be guaranteed for that purpose as long as GFP is expressed in your plant material. The only references we are currently aware of that use this antibody are:

Serio G et al. Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores. Proc Natl Acad Sci U S A 108:17337-42 (2011). IP. PubMed: 21987812

Carmena A et al. The Rap1-Rgl-Ral signaling network regulates neuroblast cortical polarity and spindle orientation. J Cell Biol 195:553-62 (2011). IP. PubMed: 22084305

The beads can appear to be translucent white when in the slurry. This is normal.Before taking out beads from the stock solution, it is important to mix the slurry well.

After the IP, when pelleting the bead-antibody-protein complex, the pellet should be clearly visible and stable so that the supernatant can be taken off without any bigger difficulties.

Please let me know how the results is looking on the WB. I am happy to help if optimisation is need.

I recommend to test-run a smaller sample first. I can recommend to use 20ulslurry (2ug of IgG cross-linked beads)with 100ug protein and check the result. If this works optimal with your samples, I then can recommend to scale it up.

Since 3mg would mean 600ul slurry, I can recommend to optimise further. Maybe 250ul slurry will be enough (this is one vial of ab69314).

I am sorry I do not have a definitive answer to this question. This has not been experimentally tested by us and we therefore cannot recommend any definitive amounts.

I can confirm that elution of the protein after IP can be done by boiling in loading buffer. This will most probably also detach some of the IP antibody from the beads (althoughantibody and beads are covalently bound)and therefore there is the risk of getting heavy and light chains of the IP antibody into the gel.

Please check with your MALDI-TOF protocol for requirements on IP and gel run. .

Replies to your questions: 1. 10ul Beads (20ul slurry) will contain 1ug IgG, each IgG (MW:150,000) can bind 2 GFP molecules (~MW:25,000 each ), therefore about 666ng of GFP should bind to these beads. Cross-linking should not interfere too much with the binding. 2. Proteins were eluted with 1% SDS 50 mM HEPES (pH 7.4) at 80oC (15 min). 3. The cross-link was carried out by a proprietary process that should minimize leaking of both IgG heavy and light chains. Typically less than 5% of the IgG content on the beads will leak. 4. By boiling samples there is a risk of having the antibody eluted too, therefore a mild eluent, such as glycine buffer, should be used to avoid this from happening.

Thank you for your inquiry. Both antibodies will work quite well actually, but we would recommend the antibody cross-linked to sepharose-beads (ab69314) simply because the lab worked with it for a much longer period of time than the antibody cross-linked to magnetic beads (ab69315). I would recommend our rabbit-anti-GFP-crosslinked to sepharose beads for the IP, then use Ab290 or Ab6556 for the WB. If the GFP concentration is really low, you may want to increase the volume of the sample by making more of it. Our IP protocol worked just as well in 1ml or 10 ml because of the high avidity of the antibody for the antigen. Incubate sample with the antibodies for 2-4 hours at 4C. Keep the amount of beads low and centrifuge thoroughly to increase beads recovery. If you incubate overnight make sure you have protease inhibitors in the lysates. I hope this information helps. Please contact me with any other questions.