c**a
发帖数: 94 | 1 就是Histone acetylation/deacetylation,蛋白放一起做in vitro assay. 有没有什么
trick啊？买来的recombinant protein都不管用，positive control都做不出来。不知
道到底
哪里出了问题。 | A******y
发帖数: 2041 | 2 Where do you get your HDACs? Also, which HDACs? The in vitro data for most
class IIa HDAC are wrong...including HDAC8. Class IIa and HDAC8 do not
deacetylate the acetylated substrate (or very little)...they do bind though.
I can give you an activity list...
Class I (Ac substrate)
HDAC3 > HDAC2 > HDAC1
Class IIa (TFA substrate)
HDAC8 > HDAC4 = HDAC5 = HDAC7 = HDAC9...
Class IIb (Ac substrate)
HDAC6 not so active but better than HDAC1
HDAC3 is at least x100 time more active than HDAC1/2 in vitro.
If you substrate doesn't have unprotected R and K, you can used it with
normal cell lysate and test your kit...the so call developer is 10mg/mL
Trypsin. | c**a
发帖数: 94 | 3 遇到高手了,讨教一下. 我用的是HDAC6, 从 Enzo 买的, 拿来做tubulin deAc, 这个在
2002的paper
中就show过了, 但是我做死都做不出来. 到底是买的protein失活了,还是反应需要特殊
的条件? 也
试过用Hela nuclear extract 加purified Histone, 也做不出来. 请高手明示. 另外,
你说的 TFA
substrate是什么?
most
though.
【在 A******y 的大作中提到】
: Where do you get your HDACs? Also, which HDACs? The in vitro data for most
: class IIa HDAC are wrong...including HDAC8. Class IIa and HDAC8 do not
: deacetylate the acetylated substrate (or very little)...they do bind though.
: I can give you an activity list...
: Class I (Ac substrate)
: HDAC3 > HDAC2 > HDAC1
: Class IIa (TFA substrate)
: HDAC8 > HDAC4 = HDAC5 = HDAC7 = HDAC9...
: Class IIb (Ac substrate)
: HDAC6 not so active but better than HDAC1
| A******y
发帖数: 2041 | 4 What's your substrate? Are you using Enzo substrate? If your hela cell
extract didn't work, don't expect the HDAC6 to work.
Check your pH...should be 8
Do the incubation at room temp first.
HDAC6 is fragile...I will get them from BPSbiosciences.
Go buy a tube of Boc-Lys(ac)-AMC from Bachem or Anaspec and make the
substrate youself in DMSO...
watch your %DMSO...more than 1% in the reaction mix will not work (yea, I
know someone say they have 5%...sure)...
If you are trying to deacetylate isolated tubulin...good luck...there are
probably acetyl-transferase somewhere in the lysate, too. |
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