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发信人: aoexy (lili), 信区: Biology
标 题: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 08:40:13 2002) WWW-POST
I run a SDS-gradient gel, then renature the target protein with glycerol. But
found the size of target protein is almost 2/3 smaller than the expected
size(deduced from it sequence)?
Is it possible that gradient Gel can change the size of protein?
Thanks very much!
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 137.132.3.10]
发信人: Protein (A Happy Guangguang), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 12:14:01 2002), 站内信件
maybe the conformation is changed?
【 在 aoexy (lili) 的大作中提到: 】
: I run a SDS-gradient gel, then renature the target protein with glycerol. But
: found the size of target protein is almost 2/3 smaller than the expected
: size(deduced from it sequence)?
: Is it possible that gradient Gel can change the size of protein?
: Thanks very much!
--
情到深处人寂寞
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.135.110.166]
发信人: leeky (leky), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 15:16:58 2002), 站内信件
After renaturing the protein, how do you determine the protein size? Gel filtration?
【 在 Protein (A Happy Guangguang) 的大作中提到: 】
: maybe the conformation is changed?
: 【 在 aoexy (lili) 的大作中提到: 】
: : I run a SDS-gradient gel, then renature the target protein with glycerol. But
: : found the size of target protein is almost 2/3 smaller than the expected
: : size(deduced from it sequence)?
: : Is it possible that gradient Gel can change the size of protein?
: : Thanks very much!
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.8.72.46]
发信人: burgen (至尊宝), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 15:28:58 2002) WWW-POST
how did you determine the protein size after renaturation?
【 在 aoexy (lili) 的大作中提到: 】
: I run a SDS-gradient gel, then renature the target protein with glycerol.
But
: found the size of target protein is almost 2/3 smaller than the expected
: size(deduced from it sequence)?
:
: Is it possible that gradient Gel can change the size of protein?
:
: Thanks very much!
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 129.93.135.210]
发信人: Protein (A Happy Guangguang), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 15:28:59 2002), 站内信件
no idea
i have not done any protein work for almost 2 years
【 在 leeky (leky) 的大作中提到: 】
: After renaturing the protein, how do you determine the protein size? Gel filtration?
: 【 在 Protein (A Happy Guangguang) 的大作中提到: 】
: : maybe the conformation is changed?
--
情到深处人寂寞
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.135.110.166]
发信人: Protein (A Happy Guangguang), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 15:30:07 2002), 站内信件
i guess you can cut the band and elute the protein and then determine the
size again
i donot know whether it is practical
you might not get too much sample after that
【 在 burgen (至尊宝) 的大作中提到: 】
: how did you determine the protein size after renaturation?
: 【 在 aoexy (lili) 的大作中提到: 】
: : I run a SDS-gradient gel, then renature the target protein with glycerol.
: But
: : found the size of target protein is almost 2/3 smaller than the expected
: : size(deduced from it sequence)?
: : Is it possible that gradient Gel can change the size of protein?
: : Thanks very much!
--
情到深处人寂寞
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.135.110.166]
发信人: Protein (A Happy Guangguang), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 15:30:48 2002), 站内信件
obviously a Yuan Zhu SDS gel will help a lot in term of collecting samples
【 在 Protein (A Happy Guangguang) 的大作中提到: 】
: i guess you can cut the band and elute the protein and then determine the
: size again
: i donot know whether it is practical
: you might not get too much sample after that
: 【 在 burgen (至尊宝) 的大作中提到: 】
: : how did you determine the protein size after renaturation?
: : But
--
情到深处人寂寞
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.135.110.166]
发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 17:15:22 2002), 站内信件
the size is not necessarily the same as the predicted size.
it is possible that modification of the polypeptide including
glycosylation, phosphorylation, even proteolysis can result
in change of the size. and it is known that adjacent patches
of charged amino acid residues can change the mobility of the
polypeptide on the SDS gel. i would recommend you try another
antibody, or it may be useful to try IP with a first one and
then do western with another one.
【 在 aoexy (lili) 的大作中提到: 】
: I run a SDS-gradient gel, then renature the target protein with glycerol. But
: found the size of target protein is almost 2/3 smaller than the expected
: size(deduced from it sequence)?
: Is it possible that gradient Gel can change the size of protein?
: Thanks very much!
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 146.186.29.87]
发信人: aoexy (lili), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 22:32:46 2002) WWW-POST
Put the gel into buffer with specific substrate because target protein can act
on this substrate can display blue colour.
【 在 leeky (leky) 的大作中提到: 】
: After renaturing the protein, how do you determine the protein size? Gel
filtration?
:
: 【 在 Protein (A Happy Guangguang) 的大作中提到: 】
: : maybe the conformation is changed?
: : 【 在 aoexy (lili) 的大作中提到: 】
: : : I run a SDS-gradient gel, then renature the target protein with
glycerol. But
: : : found the size of target protein is almost 2/3 smaller than the expected
: : : size(deduced from it sequence)?
: : : Is it possible that gradient Gel can change the size of protein?
: : : Thanks very much!
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 137.132.3.10]
发信人: aoexy (lili), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 22:35:49 2002) WWW-POST
maybe, but I run SDS gel first. So even it's conformation changed, the protein
won't change it's position in the gel becuase I didn't run it anymore after
renature.
【 在 Protein (A Happy Guangguang) 的大作中提到: 】
: maybe the conformation is changed?
: 【 在 aoexy (lili) 的大作中提到: 】
: : I run a SDS-gradient gel, then renature the target protein with glycerol.
But
: : found the size of target protein is almost 2/3 smaller than the expected
: : size(deduced from it sequence)?
: : Is it possible that gradient Gel can change the size of protein?
: : Thanks very much!
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 137.132.3.10]
发信人: aoexy (lili), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 22:53:39 2002) WWW-POST
I think glycosylation and phosphorylation only can increase the size deduced
from it's DNA sequence, right? How do I whether there is proteolysis in the
prepation and runing protien. Can you recommend some method to detect it? Did
you mean to do IP with SDS Gel and do western blot with the gel after
renature?
Thanks very much!
【 在 Marble (小石头哥哥) 的大作中提到: 】
: the size is not necessarily the same as the predicted size.
: it is possible that modification of the polypeptide including
: glycosylation, phosphorylation, even proteolysis can result
: in change of the size. and it is known that adjacent patches
: of charged amino acid residues can change the mobility of the
: polypeptide on the SDS gel. i would recommend you try another
: antibody, or it may be useful to try IP with a first one and
: then do western with another one.
:
: 【 在 aoexy (lili) 的大作中提到: 】
: : I run a SDS-gradient gel, then renature the target protein with glycerol.
But
: : found the size of target protein is almost 2/3 smaller than the expected
: : size(deduced from it sequence)?
: : Is it possible that gradient Gel can change the size of protein?
: : Thanks very much!
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 137.132.3.10]
发信人: uddi (uddi), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 23:29:39 2002) WWW-POST
another possibility, u didn't denature protain fully
in SDS when you judge the size of protain by eletrophoresis.
【 在 aoexy (lili) 的大作中提到: 】
: I think glycosylation and phosphorylation only can increase the size deduced
: from it's DNA sequence, right? How do I whether there is proteolysis in the
: prepation and runing protien. Can you recommend some method to detect it?
Did
: you mean to do IP with SDS Gel and do western blot with the gel after
: renature?
:
: Thanks very much!
: 【 在 Marble (小石头哥哥) 的大作中提到: 】
: : the size is not necessarily the same as the predicted size.
: : it is possible that modification of the polypeptide including
: : glycosylation, phosphorylation, even proteolysis can result
: : in change of the size. and it is known that adjacent patches
: : of charged amino acid residues can change the mobility of the
: : polypeptide on the SDS gel. i would recommend you try another
: : antibody, or it may be useful to try IP with a first one and
: : then do western with another one.
: :
: : 【 在 aoexy (lili) 的大作中提到: 】
: : : I run a SDS-gradient gel, then renature the target protein with
glycerol.
: But
: : : found the size of target protein is almost 2/3 smaller than the expected
: : : size(deduced from it sequence)?
: : : Is it possible that gradient Gel can change the size of protein?
: : : Thanks very much!
: :
: :
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 24.112.63.230]
发信人: applease (苹果酶), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Fri Jun 21 23:31:13 2002), 站内信件
梯度胶不用来测定蛋白大小，比较一下MARKER的迁移率就知道了。如果想确定什么带是
你需要的，只要比较一下带谱就知道了。确定大小还是跑SDS胶吧。
【 在 aoexy (lili) 的大作中提到: 】
: I run a SDS-gradient gel, then renature the target protein with glycerol. But
: found the size of target protein is almost 2/3 smaller than the expected
: size(deduced from it sequence)?
: Is it possible that gradient Gel can change the size of protein?
: Thanks very much!
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: New York ]
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 140.251.13.87]
发信人: aoexy (lili), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Sat Jun 22 00:18:27 2002) WWW-POST
this gradient gel contain SDS. The size is judged according to the marker. in
this experiment only target protein can dissect substrate, so only one
specific band appear on the gel.
【 在 applease (苹果酶) 的大作中提到: 】
: 梯度胶不用来测定蛋白大小，比较一下MARKER的迁移率就知道了。如果想确定什么带是
: 你需要的，只要比较一下带谱就知道了。确定大小还是跑SDS胶吧。
:
:
: 【 在 aoexy (lili) 的大作中提到: 】
: : I run a SDS-gradient gel, then renature the target protein with glycerol.
But
: : found the size of target protein is almost 2/3 smaller than the expected
: : size(deduced from it sequence)?
: : Is it possible that gradient Gel can change the size of protein?
: : Thanks very much!
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 137.132.3.10]
发信人: Eddy (打哪儿指哪儿), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Sat Jun 22 11:25:27 2002) WWW-POST
【 在 aoexy (lili) 的大作中提到: 】
: this gradient gel contain SDS. The size is judged according to the marker.
in
: this experiment only target protein can dissect substrate, so only one
: specific band appear on the gel.
: 【 在 applease (苹果酶) 的大作中提到: 】
: : 梯度胶不用来测定蛋白大小，比较一下MARKER的迁移率就知道了。如果想确定什么带
是
: : 你需要的，只要比较一下带谱就知道了。确定大小还是跑SDS胶吧。
: :
: :
: : 【 在 aoexy (lili) 的大作中提到: 】
: : : I run a SDS-gradient gel, then renature the target protein with
glycerol.
: But
: : : found the size of target protein is almost 2/3 smaller than the expected
: : : size(deduced from it sequence)?
: : : Is it possible that gradient Gel can change the size of protein?
: : : Thanks very much!
: :
I believe you problem is definately caused by mordification.
Mordification like phosphorylation and glycosylatio will change
the both conformation and molecular weight of the protein.
In the case of phosphorylation it's difficult to predict the
behavier of protein in SDS-PAGE because phosphorylation can
either increase or decrease protein mobility in gel, even
phospohrylations are on the same protein but on different sites.
And so does glycosylation.
For some unknown reasons SDS cannot denature protein totally as
expected, especially mordified proteins.
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 172.145.156.160]
发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: Why does the protein size change?
发信站: The unknown SPACE (Sat Jun 22 16:12:18 2002), 站内信件
i suggest you do western blot with another antibody. i do not
know what kind of antibody you are using now. if it is designed
to recognize a peptidyl epitope, it would be nicer if you can
actually use a polyclonal antibody against the whole polypeptide.
typically i would try different antibodies that recognize either
N- or C- terminus. if both of them recognize the same protein,
then proteolysis is excluded. for detection of proteolysis, i
would rather optimize the condition for isolation of protein
first and then do the blot.
do you know SREBP as well as IRE1? read those publications and
find how they know that there is proteolysis that controls its
biological activity. a question, what is your protein? and the
subcellular localization?
【 在 aoexy (lili) 的大作中提到: 】
: I think glycosylation and phosphorylation only can increase the size deduced
: from it's DNA sequence, right? How do I whether there is proteolysis in the
: prepation and runing protien. Can you recommend some method to detect it? Did
: you mean to do IP with SDS Gel and do western blot with the gel after
: renature?
: Thanks very much!
: 【 在 Marble (小石头哥哥) 的大作中提到: 】
: : the size is not necessarily the same as the predicted size.
: : it is possible that modification of the polypeptide including
: : glycosylation, phosphorylation, even proteolysis can result
: : in change of the size. and it is known that adjacent patches
: : of charged amino acid residues can change the mobility of the
: : polypeptide on the SDS gel. i would recommend you try another
: : antibody, or it may be useful to try IP with a first one and
: : then do western with another one.
: But
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 146.186.29.87]
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