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How can I decrease IgG background?


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#1 little panda

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Posted 04 April 2010 - 06:17 PM

Hi, everyone, I am a beginner of ChIP assay. In my recent experiments, I find IgG background is too high through q-PCR, and some times it even higher than antibody group. I used H23 cell line and cell number is about 1,000,000 (one 10cm plate) . I think my antibody may not very good because it has been stored at -20 cent degree for at least two years. However, I am sure that it is ChIP grade, and it is still OK for western blot. I add 2 ug antibody into chromatin. As for IgG antibody, I used a new one from santa crus. Does my poor antibody will cause this problem? How can I decrease the background?

I still have a question : To reduce IgG background, I washed Sepharose A beads three times with TSEⅡ and BufferⅢ, respectly. Does the antibody that binding with beads could be washed away because of too many wash procedure?



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℃

#2 KPDE

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Posted 05 April 2010 - 07:07 AM

Hi, everyone, I am a beginner of ChIP assay. In my recent experiments, I find IgG background is too high through q-PCR, and some times it even higher than antibody group. I used H23 cell line and cell number is about 1,000,000 (one 10cm plate) . I think my antibody may not very good because it has been stored at -20 cent degree for at least two years. However, I am sure that it is ChIP grade, and it is still OK for western blot. I add 2 ug antibody into chromatin. As for IgG antibody, I used a new one from santa crus. Does my poor antibody will cause this problem? How can I decrease the background?

I still have a question : To reduce IgG background, I washed Sepharose A beads three times with TSEⅡ and BufferⅢ, respectly. Does the antibody that binding with beads could be washed away because of too many wash procedure?



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℃



The first thing I would try is reducing the amount of chromatin that you use. Try 4X, 8X, and 16X less chromatin and see if the ratio of specific IP to IgG improves. In the past, titrating the amount of chromatin has been the most effective means of reducing background for me, which is why I always suggest it. The only reason not to try this first is if you are down near your detection limit for whatever means you are using to detect the DNA.

#3 Dukey

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Posted 05 April 2010 - 10:08 AM

Perhaps try protein A/G magnetic beads. They give VERY low background and indeed create an opposite problem - no signal at all. I like the Dynabeads from Invitrogen, they are outstanding.

#4 little panda

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Posted 06 April 2010 - 06:23 AM

Hi, everyone, I am a beginner of ChIP assay. In my recent experiments, I find IgG background is too high through q-PCR, and some times it even higher than antibody group. I used H23 cell line and cell number is about 1,000,000 (one 10cm plate) . I think my antibody may not very good because it has been stored at -20 cent degree for at least two years. However, I am sure that it is ChIP grade, and it is still OK for western blot. I add 2 ug antibody into chromatin. As for IgG antibody, I used a new one from santa crus. Does my poor antibody will cause this problem? How can I decrease the background?

I still have a question : To reduce IgG background, I washed Sepharose A beads three times with TSEⅡ and BufferⅢ, respectly. Does the antibody that binding with beads could be washed away because of too many wash procedure?



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℃



The first thing I would try is reducing the amount of chromatin that you use. Try 4X, 8X, and 16X less chromatin and see if the ratio of specific IP to IgG improves. In the past, titrating the amount of chromatin has been the most effective means of reducing background for me, which is why I always suggest it. The only reason not to try this first is if you are down near your detection limit for whatever means you are using to detect the DNA.




Thank you for your suggestion! You mean I should decrease the amount of chromatin or I should dilute it? If I reduce the amount of chromatin, I am afraid that the amount of DNA may be too small to be dected. My cell number is about 1 *10e6(one 10cm plate). Does small amount of cells will induce high IgG background?

#5 little panda

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Posted 06 April 2010 - 06:25 AM

Perhaps try protein A/G magnetic beads. They give VERY low background and indeed create an opposite problem - no signal at all. I like the Dynabeads from Invitrogen, they are outstanding.


Thank you! I will try to persuade my boss to buy some Dynabeads .

#6 Mighty Mouse

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Posted 06 April 2010 - 10:35 AM

Perhaps try protein A/G magnetic beads. They give VERY low background and indeed create an opposite problem - no signal at all. I like the Dynabeads from Invitrogen, they are outstanding.



Dukey,
Can you buy the dynabeads for ChIP from Invitrogen outside of their kits? I can't seem to find the info for ordering just the beads on their website. I've been using Millipore Protein A Magnetic Beads, but this company is a pain in the neck; the beads have been on back order for 6 weeks and I'm getting tired of dealing with them.

Thanks

MM
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#7 Dukey

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Posted 06 April 2010 - 02:39 PM

Perhaps try protein A/G magnetic beads. They give VERY low background and indeed create an opposite problem - no signal at all. I like the Dynabeads from Invitrogen, they are outstanding.



Dukey,
Can you buy the dynabeads for ChIP from Invitrogen outside of their kits? I can't seem to find the info for ordering just the beads on their website. I've been using Millipore Protein A Magnetic Beads, but this company is a pain in the neck; the beads have been on back order for 6 weeks and I'm getting tired of dealing with them.

Thanks

MM


Funny you should ask. I have been on them about splitting the kit into more affordable chunks and specifically asked them this question. Here is their response:

"Thank you for contacting Invitrogen Technical Support.

The beads In the Magnify ChIP kit is a mix of Dynabeeds Protein G and Protein A, which the ratio is proprietary and optimized for use of any antibodies in the ChIP assay. Unfortunately this mix is only sold in the Magnify ChIP kit. However, if customer would like to optimize their own protocol, they can try the Dynabeads Protein G IP kit (10006D for protein A, and 10007D for protein G). In the product manual, there is a antibody affinity table to help them to select either protein G or protein A.

I hope this information answers your questions.

Please contact us again if you have any further questions or concerns.

Regards,

Anita Yang"


You have to love that word - proprietary. From some of my contacts at Invitrogen, it appears that they are paying A LOT of royalties to someone for this kit (read: fast chip procedure) and so are really trying to make as much money as possible on it. Maybe KPDE would know something about this :lol:

#8 NemaToStella

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Posted 06 April 2010 - 07:39 PM

Yes, you can buy Dynabeads separately of any kit. We're using 10002D (protein A) and 10004D (protein G). I can only second the low background Dukey mentioned.

#9 KPDE

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Posted 06 April 2010 - 10:35 PM

Perhaps try protein A/G magnetic beads. They give VERY low background and indeed create an opposite problem - no signal at all. I like the Dynabeads from Invitrogen, they are outstanding.



Dukey,
Can you buy the dynabeads for ChIP from Invitrogen outside of their kits? I can't seem to find the info for ordering just the beads on their website. I've been using Millipore Protein A Magnetic Beads, but this company is a pain in the neck; the beads have been on back order for 6 weeks and I'm getting tired of dealing with them.

Thanks

MM


Funny you should ask. I have been on them about splitting the kit into more affordable chunks and specifically asked them this question. Here is their response:

"Thank you for contacting Invitrogen Technical Support.

The beads In the Magnify ChIP kit is a mix of Dynabeeds Protein G and Protein A, which the ratio is proprietary and optimized for use of any antibodies in the ChIP assay. Unfortunately this mix is only sold in the Magnify ChIP kit. However, if customer would like to optimize their own protocol, they can try the Dynabeads Protein G IP kit (10006D for protein A, and 10007D for protein G). In the product manual, there is a antibody affinity table to help them to select either protein G or protein A.

I hope this information answers your questions.

Please contact us again if you have any further questions or concerns.

Regards,

Anita Yang"


You have to love that word - proprietary. From some of my contacts at Invitrogen, it appears that they are paying A LOT of royalties to someone for this kit (read: fast chip procedure) and so are really trying to make as much money as possible on it. Maybe KPDE would know something about this :lol:


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.

#10 Mighty Mouse

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Posted 07 April 2010 - 04:57 AM

Yes, you can buy Dynabeads separately of any kit. We're using 10002D (protein A) and 10004D (protein G). I can only second the low background Dukey mentioned.


Thanks, that is exactly what I'm looking for. What do you use for elution buffer?

MM
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#11 Dukey

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Posted 07 April 2010 - 12:40 PM

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.

#12 Johnny Woods

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Posted 26 May 2010 - 10:12 AM

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℃
[/quote]

#13 Dr Teeth

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Posted 27 May 2010 - 06:31 AM

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.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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