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how to explain: high protein expression but undetectable mRNA
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how to explain: high protein expression but undetectable mRNA# Biology - 生物学
y*x
1
刚结婚,ld在办f1 to h4,据说现在要好几个月。这段时间h1b能离境吗?
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l*7
2
Hello, everyone, the gene I am working on has protein expression as detected
by western blot, but its mRNA in the same type of cells is undetectable by
real-time PCR. Do you know how to explain this? Thanks very much!
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s*s
3
保证你实验设计操作都没问题的情况下,会不会
你的蛋白比较稳定

detected
by

【在 l***7 的大作中提到】
: Hello, everyone, the gene I am working on has protein expression as detected
: by western blot, but its mRNA in the same type of cells is undetectable by
: real-time PCR. Do you know how to explain this? Thanks very much!

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o*i
4
我觉得你的realtime或者western有问题

detected
by

【在 l***7 的大作中提到】
: Hello, everyone, the gene I am working on has protein expression as detected
: by western blot, but its mRNA in the same type of cells is undetectable by
: real-time PCR. Do you know how to explain this? Thanks very much!

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f*e
5
反着有可能,但是你的顺序不可能阿。。。。蛋白都行,怎么可能gene不行啊

detected
by

【在 l***7 的大作中提到】
: Hello, everyone, the gene I am working on has protein expression as detected
: by western blot, but its mRNA in the same type of cells is undetectable by
: real-time PCR. Do you know how to explain this? Thanks very much!

avatar
w*e
6
你确定抗体特异性很好么?

detected
by

【在 l***7 的大作中提到】
: Hello, everyone, the gene I am working on has protein expression as detected
: by western blot, but its mRNA in the same type of cells is undetectable by
: real-time PCR. Do you know how to explain this? Thanks very much!

avatar
w*y
7
估计蛋白不特异

【在 f*********e 的大作中提到】
: 反着有可能,但是你的顺序不可能阿。。。。蛋白都行,怎么可能gene不行啊
:
: detected
: by

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f*e
8
什么意思?说抗体specificity差?
那打质普吧。。。。如果知道什么蛋白,一了白了

【在 w******y 的大作中提到】
: 估计蛋白不特异
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Z*g
9
搞一个表达该基因的阳性细胞和不表达该基因的阴性细胞作为对照,确定一下PCR或
western是否出问题.另外,你的样品是否经过处理(如药物),比方说,可能该处理抑制了
mRNA表达,但该蛋白比较稳定,半衰期长.
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l*7
10
谢谢大家的回复。
我想要把人的FOXD3基因克隆到一个病毒载体上。看到有的公司用HEPG2细胞来检测他
们的抗FOXD3的抗体 (http://www.scbt.com/datasheet-133588-foxd3-f-23-antibody.html),我就用HEPG2细胞的cDNA来PCR FOXD3,总得不到所要的片段 (得到一片段,但大了100多bp),即使是用real time pcr 的引物来检测cDNA,也没得到相应大小的片段。
有些细胞 (A-431, U-2 OS and U-251 MG) 检测不到FOXD3 RNA, 但能用FOXD3抗体
标记上,不知怎么解释。
RNA: http://www.proteinatlas.org/ENSG00000187140/rna
PROTEIN: http://www.proteinatlas.org/ENSG00000187140/cell
今天发现FOXD3只有一个外显子,没有内含子,所以我准备直接从基因组DNA里PCR试试。
谢谢。
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l*a
11
high GC 吧
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l*7
12
Yes, more than 70% GC. I have tried 10% DMSO or Enhancer Buffer from
Invitrogen for the PCR.
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l*a
13
10%DMSO 有点高了。你的PCR有positive control 或者说 在genomic DNA 上work吗?
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l*7
14
用10%的DMSO是网上看到的,也有人用3%-8%的DMSO。
没有positive control。这基因GC含量很高,ORF含有72%的GC。我重新设计了一对不带
酶切位点(接头)的引物,今天刚从genomic DNA里扩出了一条大小相符的带。明天重
复一下,就送去测序看看。
谢谢。
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