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western for methylated histone


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#1 jiro_killua

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Posted 22 February 2009 - 06:26 PM

I did some western recently and have no luck

I used regular lysis buffer (from Fisher) with protease inhibitors (from Sigma) and lyse mouse tissue (1ml/50mg)

And used 30ug protein lysate to run western

probe for H3K4me1 (will do me3 later as well), Ab from Abcam

couldn't get any band

Assuming the SDS-PAGE and transfer are ok (of course there could be one thousand possible problem with western)

Could it be that I did not load enough lysate? (well, 30ug is enough for regular western, but I don't know if a monomethylated H3K4 is ver "rare", so might need to load more protein)

Or, maybe something has to be added during lysis? (like when you do phosphorylated protein may need to add some extra inhibitors to prevent dephophorylation...)

Desperately need some help!!

#2 rick2000

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Posted 22 February 2009 - 11:52 PM

Maybe you should try acid histone extraction. Why are u saying " assuming that the SDS and transfer are ok"? Did you use prestained markers to check that the transfer was ok?

#3 jiro_killua

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Posted 23 February 2009 - 08:07 AM

Maybe you should try acid histone extraction. Why are u saying " assuming that the SDS and transfer are ok"? Did you use prestained markers to check that the transfer was ok?


yes, everything looks normal, like I normally do with western for other cellular proteins

just I'm not sure doing modified histone is special

I just read from Nature Protocols that people usually use acid extraction to purify histone

I have a naive question:
Why is it necessary to use acid extracted histone to do western rather than just lyse everything with SDS lysis buffer?

Edited by jiro_killua, 23 February 2009 - 09:28 AM.


#4 M&M81

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Posted 23 February 2009 - 05:07 PM

I haven't used whole cell extract for modified histone detection. I think ppl use acidic extract because this can get rid of many cytoplasmic proteins so that the histone proteins are more likely to be enriched in the protein sample. You could try using nuclear extract instead as some of my labmates are using this method and it works at some point. What I always do is that prepare S1+S2 chromatin fraction, see Methods & Materials of Hebbes TR et al, 1994, EMBO J. If you just want to do western blotting, I would stop the nucleosome preparation after getting S1+S2, H1 removal by adding NaCl is not necessary in your case. You may find that acidic extraction is much easier. Then the chromatin you get is ready for western blotting, for histone methylation detection, 5ug of S1+S2 is enough unless you find your band is really weak. Another tip for doing histone western blotting is that the transfer of histone is always poor, probably because histones are extremely positive charged, so you could add a bit of SDS in your transfer buffer but no more than 0.01%, and you could transfer longer, I always transfer 2 hours. If you still couldn't get any bands, you could try BSA as blocking agent, BSA would give you higher sensitivity but of course higher background as well.

Good luck!

#5 jiro_killua

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Posted 24 February 2009 - 07:09 AM

I haven't used whole cell extract for modified histone detection. I think ppl use acidic extract because this can get rid of many cytoplasmic proteins so that the histone proteins are more likely to be enriched in the protein sample. You could try using nuclear extract instead as some of my labmates are using this method and it works at some point. What I always do is that prepare S1+S2 chromatin fraction, see Methods & Materials of Hebbes TR et al, 1994, EMBO J. If you just want to do western blotting, I would stop the nucleosome preparation after getting S1+S2, H1 removal by adding NaCl is not necessary in your case. You may find that acidic extraction is much easier. Then the chromatin you get is ready for western blotting, for histone methylation detection, 5ug of S1+S2 is enough unless you find your band is really weak. Another tip for doing histone western blotting is that the transfer of histone is always poor, probably because histones are extremely positive charged, so you could add a bit of SDS in your transfer buffer but no more than 0.01%, and you could transfer longer, I always transfer 2 hours. If you still couldn't get any bands, you could try BSA as blocking agent, BSA would give you higher sensitivity but of course higher background as well.

Good luck!


Thanks so much!!

a few more questions:

1. if only the histones were extracted (say, by acid extraction), then how to normalize for loading? (can't use beta-actin?)

2. for transferring, will nitrocellulose be better than PVDF?

#6 M&M81

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Posted 24 February 2009 - 12:05 PM

I haven't used whole cell extract for modified histone detection. I think ppl use acidic extract because this can get rid of many cytoplasmic proteins so that the histone proteins are more likely to be enriched in the protein sample. You could try using nuclear extract instead as some of my labmates are using this method and it works at some point. What I always do is that prepare S1+S2 chromatin fraction, see Methods & Materials of Hebbes TR et al, 1994, EMBO J. If you just want to do western blotting, I would stop the nucleosome preparation after getting S1+S2, H1 removal by adding NaCl is not necessary in your case. You may find that acidic extraction is much easier. Then the chromatin you get is ready for western blotting, for histone methylation detection, 5ug of S1+S2 is enough unless you find your band is really weak. Another tip for doing histone western blotting is that the transfer of histone is always poor, probably because histones are extremely positive charged, so you could add a bit of SDS in your transfer buffer but no more than 0.01%, and you could transfer longer, I always transfer 2 hours. If you still couldn't get any bands, you could try BSA as blocking agent, BSA would give you higher sensitivity but of course higher background as well.

Good luck!


Thanks so much!!

a few more questions:

1. if only the histones were extracted (say, by acid extraction), then how to normalize for loading? (can't use beta-actin?)

2. for transferring, will nitrocellulose be better than PVDF?



1. generally, people use core histone for normalisation, like H3 or H4, if you don't want to run another blot for normalisation, you could use H4 as its size is much smaller than the other three, so that you can cut your blot (if you can separate it far from others) or detect it on the same blot after stripping your methylated histone antibody. A point to note is that if you are detecting methylated H3, don't try to strip it off and then detect H3 again. The methylated histone signal is always high so that it is very difficult to be stripped off completely, you would then just get a signal of H3 that is similar to your methylated H3 signal.

2. I always use PVDF though I think nitrocellulose is fine as well. Signal from PVDF is usually higher than that of nitrocellulose.

#7 sunil kumar

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Posted 21 July 2011 - 12:56 PM

u can comfortably do western blotting for methylaion or acetylation of histones levels using whole cell lysate, i have been doing it since many days with reproducable results. no hassles of ip or acid extraction or nuclear fractionation.

#8 StefyDE

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Posted 29 September 2012 - 12:47 AM

Hello!
I have a question, i had some problems similar to you! So the extraction o whole cell is not a problem when looking for histones? Because i did it, but i dont find the blots for the LW proteins i just find blots for the HW..and some of my labmates told me it was a mistake not doing the acid extraction insted of the normal one with lysis buffer!
Thank you




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