How can I decrease IgG background? - My IgG background is too high!!! (Apr/04/2010 )

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KPDE on Apr 6 2010, 11:35 PM said:

Oh I wish we were getting anything from Fast ChIP. The problem was that it was too easy to skirt a patent so no one wanted to bother putting up the legal fees for it. Still, I'm very happy if it made anyone's life easier.

Really? Well someone obviously is making some cash out of it. Oh well, fair play to them.

-Dukey-

HI,
Please change the IgG Ab. and wash 1-2 more times, and decrease the PCR cycles.

Would you please send you intact protocol to me?? dr.johnnywoods@gmail.com
Thnaks

This is my protocol:

1. Wash the cell monolayers 2 times with PBS
2. Prepare 1% formaldehyde: 540μl formaldehyde in 20ml PBS
3. Add 10ml 1% formaldehyde per 10cm plate, rotate gently for 10 minutes at room temperature
4. Wash the cell monolayers 2 times with ice-cold PBS, then scrape cells into 1ml ice-cold PBS and collected by centrifugation (2000rpm*4min at 4℃), remove supernatant
5. Prepare lysis buffer: 1ml lysis buffer+10μl PMSF(100*)+10μl cocktail(100*)
6. Vigorously resuspend cell pellets in 300μl lysis buffer, incubate on ice for 10 minutes.
7. Sonicate for 15s*3 times at 45% output, with 1 minutes refractory period between sonications
8. Centrifugate 12000rpm*10min at 4℃

1. Prepare dilution buffer: 1ml dilution buffer+10μl PMSF(100*)+10μl cocktail(100*)
2. Take off 20μl supernatant, and dilute to a final volume of 100μl with dilution buffer, store it at -20℃, this is the input.
For TianGen purification kit, take off 20μl supernatant, store it directly at -20℃
3. For 2 IPs, carefully take off 200μl and dilute to a final volume of 2ml with dilution buffer, then add 90μl Sepharose A beads (water:beads=1:1, mix well before add), then add 8μl salmon sperm DNA (1μg/μl). Rotate for 2h at 4℃
4. Centrifuge 1000rpm*2min at 4℃
5. Take off 1ml supernatant, add 2μg antibody; take off the other 1ml supernatant, add 2μg IgG . Rotate about 12h at 4℃
6. Add 45μl Sepharose A and 4μl salmon sperm DNA per 1ml supernatant, mix well, rotate 2h at 4℃
7. Centrifuge 1000rpm*2min at 4℃, remove supernatant
8. Gently add 1ml TSEⅠ, rotate 10min at 4℃, centrifuge 1000rpm*2min at 4℃, remove supernatant
9. Gently add 1ml TSEⅡ, rotate 10min at 4℃, centrifuge 1000rpm*2min at 4℃, remove supernatant
10. Gently add 1ml BufferⅢ, rotate 10min at 4℃, centrifuge 1000rpm*2min at 4℃, remove supernatant
11. Gently add 1ml TE, rotate 10min at 4℃, centrifuge 1000rpm*2min at 4℃, remove supernatant
12. Gently add 1ml TE, rotate 10min at 4℃, centrifuge 1000rpm*2min at 4℃, remove supernatant
13. Newly prepared elution buffer (1%SDS, 0.1M NaHCO3) (10%SDS 1ml, 0.085g NaHCO3 to 10ml)
14. Add 250μl elution buffer, rotate 15min at room temperature, centrifuge 1000rpm*2min at 4℃, collect supernatant to a new EP tube
15. Add 250μl elution buffer again, rotate 15min at room temperature, centrifuge 1000rpm*2min at 4℃, collect supernatant to the new EP tube. Now 500μl supernatant should be in the new EP tube.
16. Add 20μl 5M NaCl to the tube, incubate at least 6h at 65℃
17. Add 100μl elution buffer, rotate 15min at room temperature, centrifuge 1000rpm*2min at 4℃, collect supernatant to a new EP tube
18. Add 4μl 5M NaCl to the tube, incubate at least 6h at 65℃
19. Simultaneously, take out the input(20μl), add 80μl elution buffer and 4μl 5M NaCl, incubate at least 6h at 65℃

Since IgGs are sold as pools of normal IgGs from the host species, they are not necessarily non-specific, but only non-immune for any particular protein target. Animals do have antibodies in their blood which may contain epitopes found in your protein(s) of interest. My advise is to try a monoclonal antibody non-specific for anything in your system, such as anti-FLAG or anti-GFP as long as you are not using these in your system.