m******p 发帖数: 67 | 1 western for a 25kd protein. using serum.
but the band is clearly at 50kd.
seems dimer, but in sds-page, proteins are denatured, so should not form
dimmer. any suggestions?
thank you very much. |
C*******e 发帖数: 4348 | |
q******g 发帖数: 3858 | |
S*********s 发帖数: 304 | 4 60KD nrf2 shows up in 110KD in SDS PAGE.
nobody know the reason for many years.
所以可能性很多
【在 m******p 的大作中提到】 : western for a 25kd protein. using serum. : but the band is clearly at 50kd. : seems dimer, but in sds-page, proteins are denatured, so should not form : dimmer. any suggestions? : thank you very much.
|
a*********n 发帖数: 2526 | 5 for some dimers, SDS is not enough to fully disrupt it.
【在 m******p 的大作中提到】 : western for a 25kd protein. using serum. : but the band is clearly at 50kd. : seems dimer, but in sds-page, proteins are denatured, so should not form : dimmer. any suggestions? : thank you very much.
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t******y 发帖数: 716 | 6 ”using serum“.会不会是因为血清里的IgG Heavy Chain 污染? |
b******y 发帖数: 627 | 7 nod. I have many such examples. For instance, I have a 14mer of 10 KDa
protein. But its molecular weight is about 140 KDa on SDS-PAGE.
【在 a*********n 的大作中提到】 : for some dimers, SDS is not enough to fully disrupt it.
|
u**********d 发帖数: 573 | 8 这么神奇?什么蛋白?
【在 b******y 的大作中提到】 : nod. I have many such examples. For instance, I have a 14mer of 10 KDa : protein. But its molecular weight is about 140 KDa on SDS-PAGE.
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D******9 发帖数: 2665 | 9 add 50-100 mM DTT in your loading buffer |
k******0 发帖数: 1073 | |
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b******y 发帖数: 627 | 11 It is a yeast protein over-expressed in E. coli, in which its authentic
binding partner doesn't exist.
【在 u**********d 的大作中提到】 : 这么神奇?什么蛋白?
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s*******e 发帖数: 1010 | 12 最大的可能性就是个SDS抗性的二聚体。
SDS-resistant的蛋白多聚体挺多的,老麦的KcsA就是个例子。 |
m******p 发帖数: 67 | 13 have not tried urea. will try it.
have tried increasing dtt, but not that high. will increase even high.
modification is possible. will try a kit that removes glycosilation.
thanks guys. hopefully will update you with new results. |
a*******a 发帖数: 4233 | 14 可能有修饰,也可能是蛋白自身交联
前阵子science上的jmjd6就是自身交联,大小是理论值的2倍 sds page破不开 |
g*********5 发帖数: 2533 | 15 68kDa.
and sometime 100kDa, sometime 110kDa.
I am crazy with this one.
and most commercial antibodies do not work...
do you have a good one for mouse tissue?
(no H300)
【在 S*********s 的大作中提到】 : 60KD nrf2 shows up in 110KD in SDS PAGE. : nobody know the reason for many years. : 所以可能性很多
|
S*********s 发帖数: 304 | 16 Because in the past 10 years, people thought that 68KD is the right one.
H300 works at 1:500 to 1:1000.
That's the only one worked in my hand.
【在 g*********5 的大作中提到】 : 68kDa. : and sometime 100kDa, sometime 110kDa. : I am crazy with this one. : and most commercial antibodies do not work... : do you have a good one for mouse tissue? : (no H300)
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m******p 发帖数: 67 | 17 then, how did people know the band at 110kd is jmjd6? |
H*g 发帖数: 2333 | 18 How did u boil the sample before loading on SDS-PAGE?
Do u think if this step could affect the molecular weight of the observed
band(s)? |
m******p 发帖数: 67 | 19 i boiled it for 5 min.
a website says no boiling can remove dimer, so i tried no boilding, but
still the same result. |
C*******I 发帖数: 151 | 20 有时SDS不足以解开oligomers。多加些SDS(1.5%)和beta-mercaptoethanol(4%)。然
后在90C加热10分钟试试。
【在 m******p 的大作中提到】 : western for a 25kd protein. using serum. : but the band is clearly at 50kd. : seems dimer, but in sds-page, proteins are denatured, so should not form : dimmer. any suggestions? : thank you very much.
|
f******9 发帖数: 39 | 21 How about -S-S- linked dimer? |