FPLC: Gel Filtraito - Running Standards, Triton X-100, Mitochondria on a column (Feb/02/2007 )
Hi anyone wise in FPLC (since I am not):
With our Superdex 200 from Amersham, we ran into some pressure increases that exceeded its maximum. After NaOH cleaning it went back to normal (more or less), except for some peak tailing.
However, when I attempted to run some Sigma standards as a mixture, the void volume, or first elution, came after many, many, column volumes, rather than 1/3 column volume. I ran it in 50mM Tris pH 7.5, 300mM NaCl, 5% Glycerol. Immediately, I washed with water, then 1M NaOH, including through the sample loop. This was done until the UV280 was ~0. Then I hit it with water, then 20% EtOH. It's been in 20% EtOH ever since. Now, I want to do the standards again, feeling that I either loaded too much, the column was not clean, and/or the sample loop was not clean. I want to use 50mM Tris pH 7.5, 300mM NaCl, 5% Glycerol, adn 0.1% Triton X-100. In fact, this will be the running buffer that I will use for separating proteins from my cellular fractions. Does anyone think my column is compromised, such as crushed or whatever? I guess I could run acetone to check efficiency, but I'm a little hasty and just want to get to the standards and finally have a reference for my purifications.
What about 0.1% TX-100, is it troublesome? I've read that others have used it.
Lastly, has anyone put mitochondrial fractions on a GF or ion exchange column. Do you have to lyse the mitochondria or does conventional mitochondrial fractionation lyse them open? My concern is about putting so much lipid on a resin.
Thanks for anyone's advice in advance
TX-100 has a phenol group, so it may contribute to a high OD280. There is a reduced form of TX-100 that has low OD280 reading that you can use as an alternative.
Also, try to run each individual protein standard separately, instead of a mixture of them, because some may form aggregates when mixed together.
Be sure to centrifuge/filtrate your sample before loading it.
With our Superdex 200 from Amersham, we ran into some pressure increases that exceeded its maximum. After NaOH cleaning it went back to normal (more or less), except for some peak tailing.
However, when I attempted to run some Sigma standards as a mixture, the void volume, or first elution, came after many, many, column volumes, rather than 1/3 column volume. I ran it in 50mM Tris pH 7.5, 300mM NaCl, 5% Glycerol. Immediately, I washed with water, then 1M NaOH, including through the sample loop. This was done until the UV280 was ~0. Then I hit it with water, then 20% EtOH. It's been in 20% EtOH ever since. Now, I want to do the standards again, feeling that I either loaded too much, the column was not clean, and/or the sample loop was not clean. I want to use 50mM Tris pH 7.5, 300mM NaCl, 5% Glycerol, adn 0.1% Triton X-100. In fact, this will be the running buffer that I will use for separating proteins from my cellular fractions. Does anyone think my column is compromised, such as crushed or whatever? I guess I could run acetone to check efficiency, but I'm a little hasty and just want to get to the standards and finally have a reference for my purifications.
What about 0.1% TX-100, is it troublesome? I've read that others have used it.
Lastly, has anyone put mitochondrial fractions on a GF or ion exchange column. Do you have to lyse the mitochondria or does conventional mitochondrial fractionation lyse them open? My concern is about putting so much lipid on a resin.
Thanks for anyone's advice in advance
if your column has get overpressure, there may be some beads crushed which will explain the unusual retention; with blue dextran you can monitor if there is a consistent flow over the whole column or if there are zones of higher retention; try to eliminate defect material, and refill the column
Also, try to run each individual protein standard separately, instead of a mixture of them, because some may form aggregates when mixed together.
Be sure to centrifuge/filtrate your sample before loading it.
Thanks genehunter.
I was thinking that since I will be analyzing mixture I should analyze standards as a mixture. Does it matter? If not, then I will do as you suggested and run the standards individually.
DOCDTT