S******e
发帖数: 393 | 1 一个蛋白有没有可能有多个binding sites 与另一个蛋白binding？
我将蛋白A分成4段(no overlap)去做与B的binding,结果显示只有蛋白A的C端那个fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
还是我IP有问题？ (A的每个fragments都约70kD以上)
顺便再问一下大家做co-IP都用多少总蛋白以及binding多久？
非常感谢！ | b******e
发帖数: 88 | 2 完全可能，非常常见，如果能做定量的话还可以测一下各个fragment之间有没有
competing或者帮助binding的事情
后面那个问题不懂
fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
【在 S******e 的大作中提到】
: 一个蛋白有没有可能有多个binding sites 与另一个蛋白binding？
: 我将蛋白A分成4段(no overlap)去做与B的binding,结果显示只有蛋白A的C端那个fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
: 还是我IP有问题？ (A的每个fragments都约70kD以上)
: 顺便再问一下大家做co-IP都用多少总蛋白以及binding多久？
: 非常感谢！
| S******e
发帖数: 393 | 3 Thanks a lot!
有机会能否告知相关reference paper,
我看的paper不多，没注意过这种情况。
也不知如何定量。
多谢！
【在 b******e 的大作中提到】
: 完全可能，非常常见，如果能做定量的话还可以测一下各个fragment之间有没有
: competing或者帮助binding的事情
: 后面那个问题不懂
:
: fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
| s*********y
发帖数: 387 | 4 co ask.
fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
【在 S******e 的大作中提到】
: 一个蛋白有没有可能有多个binding sites 与另一个蛋白binding？
: 我将蛋白A分成4段(no overlap)去做与B的binding,结果显示只有蛋白A的C端那个fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
: 还是我IP有问题？ (A的每个fragments都约70kD以上)
: 顺便再问一下大家做co-IP都用多少总蛋白以及binding多久？
: 非常感谢！
| C*********m
发帖数: 213 | 5 Further insight into substrate recognition by USP7: structural and
biochemical analysis of the HdmX and Hdm2 interactions with USP7. Journal of
molecular biology 2010
【在 S******e 的大作中提到】
: Thanks a lot!
: 有机会能否告知相关reference paper,
: 我看的paper不多，没注意过这种情况。
: 也不知如何定量。
: 多谢！
| S******e
发帖数: 393 | 6 谢谢你给这篇paper,
我好好看一下，
希望我的结果是对的，
而不是IP的问题。
of
【在 C*********m 的大作中提到】
: Further insight into substrate recognition by USP7: structural and
: biochemical analysis of the HdmX and Hdm2 interactions with USP7. Journal of
: molecular biology 2010
| H*g
发帖数: 2333 | 7 Just curious, how do you know each fragment of protein A maintains the same
tertiary structure as in their endogenous uncut state? If not 100% sure
about this, how do you know any binding information you got from IP is true
or biological meaningful? Why not do mutations/substitutions?
fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
【在 S******e 的大作中提到】
: 一个蛋白有没有可能有多个binding sites 与另一个蛋白binding？
: 我将蛋白A分成4段(no overlap)去做与B的binding,结果显示只有蛋白A的C端那个fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
: 还是我IP有问题？ (A的每个fragments都约70kD以上)
: 顺便再问一下大家做co-IP都用多少总蛋白以及binding多久？
: 非常感谢！
| S******e
发帖数: 393 | 8 谢谢你的问题，我想这确实存在，但目前也有paper这么做的，
对于我来说我也很想能找到两个蛋白之间的binding site，
但不知道有什么好的方法去做。
不知你说的muatations/substitutions是否指用bioinformatics等手段
预测可能的binding sites,然后mutate后再做IP，
没有这方面的基础，是不是工作量也很大，尤其对于
几百kD的蛋白来说。
same
true
【在 H*g 的大作中提到】
: Just curious, how do you know each fragment of protein A maintains the same
: tertiary structure as in their endogenous uncut state? If not 100% sure
: about this, how do you know any binding information you got from IP is true
: or biological meaningful? Why not do mutations/substitutions?
:
: fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
| b******y
发帖数: 627 | 9 It is common to divide a huge protein into a few fragments based its domain
structure and use these fragments to narrow down its binding site with
another protein. There are no other obvious ways to do it. Of course, by
doing so, you may not find the robust binding if it is provided through
cooperativity among more than one binding surfaces. This scenario is not
uncommon in biology.
My guess is that your protein A is the extracellular portion of a protein.
If so, I am sure there are a lot of well defined, small modular domains.
Your current way of doing is, again, of no problem. However, normally
interactions involving extracellular proteins are of relatively low affinity
. Therefore, I am curious how strong the bindings between fragments 1, 2, or
3 and protein B are, i.e. how stringent your IP conditions are.
The same question holds if my guess is wrong. I would make sure the bindings
are not non-specific. | s**a
发帖数: 293 | 10 完全有可能啊……而且跟片段大小关系不大吧
fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
【在 S******e 的大作中提到】
: 一个蛋白有没有可能有多个binding sites 与另一个蛋白binding？
: 我将蛋白A分成4段(no overlap)去做与B的binding,结果显示只有蛋白A的C端那个fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
: 还是我IP有问题？ (A的每个fragments都约70kD以上)
: 顺便再问一下大家做co-IP都用多少总蛋白以及binding多久？
: 非常感谢！
| | | s*********y
发帖数: 387 | 11 do not understand.
A-B-C-D, four domain proteins, and when doing interaction studies,
A, B, D, can associate with protein X, except C domain fragment?
How to explain the binding mechanism?
【在 s**a 的大作中提到】
: 完全有可能啊……而且跟片段大小关系不大吧
:
: fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
| r***e
发帖数: 2539 | 12 高级结构？
【在 s*********y 的大作中提到】
: do not understand.
: A-B-C-D, four domain proteins, and when doing interaction studies,
: A, B, D, can associate with protein X, except C domain fragment?
: How to explain the binding mechanism?
| b******y
发帖数: 627 | 13 In biology, there are a lot of multivalent/multivalent interactions from
extracellular scafolding via protein/sugar interactions, intracellular
signaling pathways via protein/protein interactions, to RNA metabolism via
protein/RNA interactions to DNA packaging and modifications via protein/DNA
interactions. For example, protein A contains a few similar domains, each of
which can bind protein B. Very often, protein B contains a few domains that
can binding those in protein A. Now protein A and B form multivalent/
multivalent interactions.
You can certainly call this 高级结构, which is beyond ternary structure.
However, I am not implying that the interactions described by LZ are
specific and functional relevant. More work is needed in this case, in my
opinion. | s**a
发帖数: 293 | 14 他说的应该是c terminal……可能尾部的片段对binding不太重要，我就做过这样的
constructs，从c端几乎删掉蛋白全长的一半，相互作用基本不变。
顺便搭车求co-IP需要的蛋白浓度
【在 s*********y 的大作中提到】
: do not understand.
: A-B-C-D, four domain proteins, and when doing interaction studies,
: A, B, D, can associate with protein X, except C domain fragment?
: How to explain the binding mechanism?
| b******y
发帖数: 627 | 15 In my opinion, the required protein concentrations in co-IP (using
recombinant or semi-pure proteins) experiments are dictated by the
sensitivity of your detection method and the binding affinity. There are no
gold standard. Generally, you have to try around.
I am suggesting some reasonable start here. For high affinity binding events
(tens of nM or tighter), a few micrograms of protein are sufficient. If you
load everything into one or two lanes, coomassie blue staining should pick
it up easily. You can dial down the loading amount per lane if you are using
silver stain or western blotting. For relatively low binding affinity
events (hundreds of nM to tens of uM), I will start with 20 micrograms of
both partners to see what you can get. Then adjust according to outcome. | S******e
发帖数: 393 | 16 谢谢！
我做了两次，基本是一致的结果，目前还在做。
用的是sigma的M2 flag agarose beads,
600-1000ug总蛋白做IP，pre-clearance with IgG beads for 3hrs,
add flag beads, 4度rotate for 5hrs,
wash 3 times with lysis buffer,
then wash once with high salt(500mM NaCl) lysis buffer,
final wash with TBS,
SDS sample buffer elute(下次准备用flag peptide elute)
还请多指教。
domain
affinity
【在 b******y 的大作中提到】
: It is common to divide a huge protein into a few fragments based its domain
: structure and use these fragments to narrow down its binding site with
: another protein. There are no other obvious ways to do it. Of course, by
: doing so, you may not find the robust binding if it is provided through
: cooperativity among more than one binding surfaces. This scenario is not
: uncommon in biology.
: My guess is that your protein A is the extracellular portion of a protein.
: If so, I am sure there are a lot of well defined, small modular domains.
: Your current way of doing is, again, of no problem. However, normally
: interactions involving extracellular proteins are of relatively low affinity
| S******e
发帖数: 393 | 17 对，是c-terminal fragment, 大约是全长的1/3
【在 s**a 的大作中提到】
: 他说的应该是c terminal……可能尾部的片段对binding不太重要，我就做过这样的
: constructs，从c端几乎删掉蛋白全长的一半，相互作用基本不变。
: 顺便搭车求co-IP需要的蛋白浓度
| S******e
发帖数: 393 | 18 谢谢，目前还在做，看来还真有可能，
我那个B蛋白也是用fragment mapping的方法，
然后确定了某一区段是与A binding 需要的
【在 s**a 的大作中提到】
: 完全有可能啊……而且跟片段大小关系不大吧
:
: fragment与B没有binding, 其它3个fragments都与B有binding,这可能吗？
| t*****P
发帖数: 36 | 19 IgG control?-negative control--Mock plasmid- | S******e
发帖数: 393 | 20 忘了说了，
negative control是transfect with vector expressing double tag
【在 t*****P 的大作中提到】
: IgG control?-negative control--Mock plasmid-
| b******y
发帖数: 627 | 21
【在 S******e 的大作中提到】
: 谢谢！
: 我做了两次，基本是一致的结果，目前还在做。
: 用的是sigma的M2 flag agarose beads,
: 600-1000ug总蛋白做IP，pre-clearance with IgG beads for 3hrs,
: add flag beads, 4度rotate for 5hrs,
: wash 3 times with lysis buffer,
: then wash once with high salt(500mM NaCl) lysis buffer,
: final wash with TBS,
: SDS sample buffer elute(下次准备用flag peptide elute)
: 还请多指教。
| b******y
发帖数: 627 | 22 High salt can help eliminating non-specific interactions through salt
bridges. Adding some detergent, say 0.02% NP-40 or something alike, can help
eliminating non-specific interactions through hydrophobic interactions.
If affordable, I would wash three-five times with high salt with detergent
buffer.
【在 S******e 的大作中提到】
: 谢谢！
: 我做了两次，基本是一致的结果，目前还在做。
: 用的是sigma的M2 flag agarose beads,
: 600-1000ug总蛋白做IP，pre-clearance with IgG beads for 3hrs,
: add flag beads, 4度rotate for 5hrs,
: wash 3 times with lysis buffer,
: then wash once with high salt(500mM NaCl) lysis buffer,
: final wash with TBS,
: SDS sample buffer elute(下次准备用flag peptide elute)
: 还请多指教。
|
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