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.
s*c
3 楼
grocery, supermarket
C*e
4 楼
加urea呢
T*t
5 楼
To be eligible for this offer, you must be the named recipient of this invitation. 是不是没收到invitation的就不能注册?
q*g
6 楼
protein modification?
j*o
7 楼
是的.这是TARGETED OFFER
【在 T**********t 的大作中提到】 : To be eligible for this offer, you must be the named recipient of this : invitation. 是不是没收到invitation的就不能注册?
S*s
8 楼
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
9 楼
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
10 楼
”using serum“.会不会是因为血清里的IgG Heavy Chain 污染?
b*y
11 楼
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
12 楼
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
13 楼
add 50-100 mM DTT in your loading buffer
k*0
14 楼
样品加高浓度的DTT或TCEP没有?
b*y
15 楼
It is a yeast protein over-expressed in E. coli, in which its authentic binding partner doesn't exist.
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.
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
20 楼
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
21 楼
then, how did people know the band at 110kd is jmjd6?
H*g
22 楼
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
23 楼
i boiled it for 5 min. a website says no boiling can remove dimer, so i tried no boilding, but still the same result.
【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.