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[其他] 为什么计算的蛋白分子量和抗体说明书杂出的分子量不一致

疑惑:
一直有个问题比较困扰,特来求教
我目前在做PS1蛋白的WB,我根据PS1的氨基酸构成用DNAMAN计算的分子量为55KD左右,但是买来的抗体说明书明确指出目的条带在32KD,请问为何?

参考一:
是不是人家用的32kd的免疫原制作的抗体,而你计算出来的是全蛋白的mw。

参考二:
你应该使用的下面这个蛋白吧
Protein names Recommended name:
Presenilin-1
Short name=PS-1
EC=3.4.23.-
Alternative name:
Protein S182
Cleaved into the following 3 chains:
1- Recommended name:
Presenilin-1 NTF subunit
2- Recommended name:
Presenilin-1 CTF subunit
3- Recommended name:
Presenilin-1 CTF12
Short name=PS1-CTF12
Gene names Name: PSEN1
Synonyms: AD3, PS1, PSNL1
他有下面几种isoform:
Sequence Length Mass (Da) Isoform 1 (I-467)
467 52,668
10 20 30 40 50 60
MTELPAPLSY FQNAQMSEDN HLSNTVRSQN DNRERQEHND RRSLGHPEPL SNGRPQGNSR
70 80 90 100 110 120
QVVEQDEEED EELTLKYGAK HVIMLFVPVT LCMVVVVATI KSVSFYTRKD GQLIYTPFTE
130 140 150 160 170 180
DTETVGQRAL HSILNAAIMI SVIVVMTILL VVLYKYRCYK VIHAWLIISS LLLLFFFSFI
190 200 210 220 230 240
YLGEVFKTYN VAVDYITVAL LIWNFGVVGM ISIHWKGPLR LQQAYLIMIS ALMALVFIKY
250 260 270 280 290 300
LPEWTAWLIL AVISVYDLVA VLCPKGPLRM LVETAQERNE TLFPALIYSS TMVWLVNMAE
310 320 330 340 350 360
GDPEAQRRVS KNSKYNAEST ERESQDTVAE NDDGGFSEEW EAQRDSHLGP HRSTPESRAA
370 380 390 400 410 420
VQELSSSILA GEDPEERGVK LGLGDFIFYS VLVGKASATA SGDWNTTIAC FVAILIGLCL
430 440 450 460
TLLLLAIFKK ALPALPISIT FGLVFYFATD YLVQPFMDQL AFHQFYI
Isoform 2 (I-463). 463 52,197
10 20 30 40 50 60
MTELPAPLSY FQNAQMSEDN HLSNTNDNRE RQEHNDRRSL GHPEPLSNGR PQGNSRQVVE
70 80 90 100 110 120
QDEEEDEELT LKYGAKHVIM LFVPVTLCMV VVVATIKSVS FYTRKDGQLI YTPFTEDTET
130 140 150 160 170 180
VGQRALHSIL NAAIMISVIV VMTILLVVLY KYRCYKVIHA WLIISSLLLL FFFSFIYLGE
190 200 210 220 230 240
VFKTYNVAVD YITVALLIWN FGVVGMISIH WKGPLRLQQA YLIMISALMA LVFIKYLPEW
250 260 270 280 290 300
TAWLILAVIS VYDLVAVLCP KGPLRMLVET AQERNETLFP ALIYSSTMVW LVNMAEGDPE
310 320 330 340 350 360
AQRRVSKNSK YNAESTERES QDTVAENDDG GFSEEWEAQR DSHLGPHRST PESRAAVQEL
370 380 390 400 410 420
SSSILAGEDP EERGVKLGLG DFIFYSVLVG KASATASGDW NTTIACFVAI LIGLCLTLLL
430 440 450 460
LAIFKKALPA LPISITFGLV FYFATDYLVQ PFMDQLAFHQ FYI
Isoform 3 (I-374). 374 42,287
10 20 30 40 50 60
MTELPAPLSY FQNAQMSEDN HLSNTNDNRE RQEHNDRRSL GHPEPLSNGR PQGNSRQVVE
70 80 90 100 110 120
QDEEEDEELT LKYGAKHVIM LFVPVTLCMV VVVATIKSVS FYTRKDGQLI YTPFTEDTET
130 140 150 160 170 180
VGQRALHSIL NAAIMISVIV VMTILLVVLY KYRCYKVIHA WLIISSLLLL FFFSFIYLGE
190 200 210 220 230 240
VFKTYNVAVD YITVALLIWN FGVVGMISIH WKGPLRLQQA YLIMISALMA LVFIKYLPEW
250 260 270 280 290 300
TAWLILAVIS VYDLVAVLCP KGPLRMLVET AQERNETLFP ALIYSSTMVW LVNMAEGDPE
310 320 330 340 350 360
AQRRVSKNSK YNAERACLPP AAINLLSIAP MAPRLFMPKG ACRPTAQKGS HKTLLQRMMM
370
AGSVRNGKPR GTVI
Isoform 4 1 84 21,073
10 20 30 40 50 60
MTELPAPLSY FQNAQMSEDN HLSNTVRSQN DNRERQEHND RRSLGHPEPL SNGRPQGNSR
70 80 90 100 110 120
QVVEQDEEED EELTLKYGAK HVIMLFVPVT LCMVVVVATI KSVSFYTRKD GQLIYTPFTE
130 140 150 160 170 180
DTETVGQRAL HSILNAAIMI SVIVVMTILL VVLYKYRCYK VSMRHRSLLS TLFFLWLGIL
VTVT
Isoform 5. 378 42,757
10 20 30 40 50 60
MTELPAPLSY FQNAQMSEDN HLSNTVRSQN DNRERQEHND RRSLGHPEPL SNGRPQGNSR
70 80 90 100 110 120
QVVEQDEEED EELTLKYGAK HVIMLFVPVT LCMVVVVATI KSVSFYTRKD GQLIYTPFTE
130 140 150 160 170 180
DTETVGQRAL HSILNAAIMI SVIVVMTILL VVLYKYRCYK VIHAWLIISS LLLLFFFSFI
190 200 210 220 230 240
YLGEVFKTYN VAVDYITVAL LIWNFGVVGM ISIHWKGPLR LQQAYLIMIS ALMALVFIKY
250 260 270 280 290 300
LPEWTAWLIL AVISVYDLVA VLCPKGPLRM LVETAQERNE TLFPALIYSS TMVWLVNMAE
310 320 330 340 350 360
GDPEAQRRVS KNSKYNAERA CLPPAAINLL SIAPMAPRLF MPKGACRPTA QKGSHKTLLQ
370
RMMMAGSVRN GKPRGTVI
Isoform 6. 409 46,328
10 20 30 40 50 60
MTELPAPLSY FQNAQMSEDN HLSNTVRSQN DNRERQEHND RRSLGHPEPL SNGRPQGNSR
70 80 90 100 110 120
QVVEQDEEED EELTLKYGAK HVIMLFVPVT LCMVVVVATI KSVSFYTRKD GQLIYTPFTE
130 140 150 160 170 180
DTETVGQRAL HSILNAAIMI SVIVVMTILL VVLYKYRCYK VIHAWLIISS LLLLFFFSFI
190 200 210 220 230 240
YLGEVFKTYN VAVDYITVAL LIWNFGVVGM ISIHWKGPLR LQQAYLIMIS ALMALVFIKY
250 260 270 280 290 300
LPEWTAWLIL AVISVYDLVA VLCPKGPLRM LVETAQERNE TLFPALIYSS TMVWLVNMAE
310 320 330 340 350 360
GDPEAQRRVS KNSKYNAERG VKLGLGDFIF YSVLVGKASA TASGDWNTTI ACFVAILIGL
370 380 390 400
CLTLLLLAIF KKALPALPIS ITFGLVFYFA TDYLVQPFMD QLAFHQFYI
你算出的55KD是isoform1。上面几种isoform都没有抗体说明书上指出的32KD,那就还有一种可能,即客户使用重组蛋白而不是天然蛋白作为检测蛋白。
如果你能给出更多的信息,我们才能给出更进一步的说明。

参考三:
下面是我复制的说明书上的一段,他所说的并不是重组蛋白,Presenilin-1, human. By Western blot the antibody recognizes a predominant 32 kDa polypeptide in a variety of samples, including PC12 cells transfected with human PS1 complementary DNA, brain biopsy specimens from demented patients, and postmortem samples of frontal neucortex from Familial Alzheimer's Disease cases, late-onset Alzheimer's disease cases, and cases of other degenerative disorders. Immunohistochemical studies of control brains revealed that PS1 is expressed primarily in neurons, with the protein localizing in the soma and dendritic processes. In contrast in FAD and sporadic Alzheimer's disease cases, PS1 immunoreactivity was present in the neuritic component of senile plaques as well as in neurofibrillary tangles. Localization of PS1 immunoreactivity in familial and sporadic Alzheimer's disease suggest that genetically heterogeneous forms of the disease share a common pathophysiology involving PS1 protein.

参考四:
这个产品你是买的millipore的吧。我上网站看了一下,无图片,并且目的条带位置“Recognizes Presenilin-1, human. By Western blot the antibody recognizes a predominant 32 kDa polypeptide in a variety of samples”与蛋白大小“SIZE: 467 amino acids; 52668 Da ”相差很大。在一般情况下我们尽量不要购买这种产品。

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meibo520 离线

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只看该作者, 1 发表于: 2016-09-30
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meibo520 离线

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只看该作者, 2 发表于: 2016-09-30
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