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.
|
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.
|
D******9
发帖数: 2665 | 9 add 50-100 mM DTT in your loading buffer |
k******0
发帖数: 1073 | |
|
|
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？
|
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)
|
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? |
