C******8
发帖数: 602 | 1 听版上人说做CoImmunoprecipitaion用magnetic beads可以减少nonspecific
手里刚好有active motif ChIP kit里的magnetic protein G beads
我可不可以用这个beads conjugate antibody，然后去拽cell lysate的antigen呀。心
里想差别也就是ChIP里protein先crosslink了DNA嘛。。。
washbuffer还有elution buffer有什么要注意的么？Elution难道直接加sds sample
loading buffer煮一煮，dissociate下来protein到buffer里然后load？
如果新买其它beads还得等几天，试试可不可以呀？有没有前人经验。。。 | n******5
发帖数: 21 | 2 我觉得M-beads, 主要是方便，和 minimized carry-over therefore increase
recovery. if you want to minize non-specific interactions, try follow some
naked beads to absorb them.
但是缺点就是 binding capacity 很低。
Wash buffer:建议加一些non-ionic detergent, like Triton100
Elution buffer: SDS+boil will get everything off, but all in denatured form,
it is perfectly fine if you directly go on to SDSPAGE.
if you want to maintain functional activity, I would suggest using low pH
elution (Try pH2.0 glycine, use Tris-pH8 to neutralize).
Try this Kit. it includes all the buffers you needed.
https://products.invitrogen.com/ivgn/product/10007D | r******y
发帖数: 21907 | 3 不错，我也需要m beads信息。。。
form,
【在 n******5 的大作中提到】
: 我觉得M-beads, 主要是方便，和 minimized carry-over therefore increase
: recovery. if you want to minize non-specific interactions, try follow some
: naked beads to absorb them.
: 但是缺点就是 binding capacity 很低。
: Wash buffer:建议加一些non-ionic detergent, like Triton100
: Elution buffer: SDS+boil will get everything off, but all in denatured form,
: it is perfectly fine if you directly go on to SDSPAGE.
: if you want to maintain functional activity, I would suggest using low pH
: elution (Try pH2.0 glycine, use Tris-pH8 to neutralize).
: Try this Kit. it includes all the buffers you needed.
| z****g
发帖数: 3340 | 4 don't need any conjugation with beads. just incubate the ab with magnetic
beads and then your cell extract.
good luck.
【在 C******8 的大作中提到】
: 听版上人说做CoImmunoprecipitaion用magnetic beads可以减少nonspecific
: 手里刚好有active motif ChIP kit里的magnetic protein G beads
: 我可不可以用这个beads conjugate antibody，然后去拽cell lysate的antigen呀。心
: 里想差别也就是ChIP里protein先crosslink了DNA嘛。。。
: washbuffer还有elution buffer有什么要注意的么？Elution难道直接加sds sample
: loading buffer煮一煮，dissociate下来protein到buffer里然后load？
: 如果新买其它beads还得等几天，试试可不可以呀？有没有前人经验。。。
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