j*****a
发帖数: 92 | 1 用size exclusive column (Sephacryl 100, buffer Tris-HCl (pH 8.5) 5% glycerol
)分离 ionic exchange column pools (desired protein 35 kda).所有的蛋白在死体
积同时下来，完全没有分离.请问怎么办？谢谢. |
m**z
发帖数: 787 | 2 your sample loading volume and column volume? you said all proteins, that
means all proteins including your target protein and the impurities? Do you
have any salt in your running buffer?
glycerol
【在 j*****a 的大作中提到】
: 用size exclusive column (Sephacryl 100, buffer Tris-HCl (pH 8.5) 5% glycerol
: )分离 ionic exchange column pools (desired protein 35 kda).所有的蛋白在死体
: 积同时下来，完全没有分离.请问怎么办？谢谢.
|
a***e
发帖数: 1010 | 3 run a control, such as 溴芬蓝 & dextran blue first. |
j*****a
发帖数: 92 | 4 your sample loading volume: 0.5 mL
column volume 50 mL
you said all proteins, that
means all proteins including your target protein: Yes
and the impurities?
Do you have any salt in your running buffer? no
Thanks |
m**z
发帖数: 787 | 5 could be proteins aggregates/precipitates during running due to no salt?
agree with the previous reply that some control/standard may be a good idea
as well
【在 j*****a 的大作中提到】
: your sample loading volume: 0.5 mL
: column volume 50 mL
: you said all proteins, that
: means all proteins including your target protein: Yes
: and the impurities?
: Do you have any salt in your running buffer? no
: Thanks
|
I*****y
发帖数: 6402 | 6 what sizing column did you use? I guess your proteins of interest have
aggregated without salt or DTT? The aggregated proteins behave like a huge proteins
that you find in the first few fractions.
glycerol
【在 j*****a 的大作中提到】
: 用size exclusive column (Sephacryl 100, buffer Tris-HCl (pH 8.5) 5% glycerol
: )分离 ionic exchange column pools (desired protein 35 kda).所有的蛋白在死体
: 积同时下来，完全没有分离.请问怎么办？谢谢.
|
y*****1
发帖数: 73 | 7 what's the PI of your protein?
If it is close to 8.5, change the pH |
s********l
发帖数: 1195 | 8 do a control first to make sure column is fine.
then it could be your protein is too small for this kinda beads, if the
protein is not aggregated.
glycerol
【在 j*****a 的大作中提到】
: 用size exclusive column (Sephacryl 100, buffer Tris-HCl (pH 8.5) 5% glycerol
: )分离 ionic exchange column pools (desired protein 35 kda).所有的蛋白在死体
: 积同时下来，完全没有分离.请问怎么办？谢谢.
|
