Sephadex G-50 gel filtration chromatography - (Feb/15/2008 )
hi guys, has anyone tried to run myoglobin on a Sephadex G-50 gel filtration chromatography column before? i can't seem to detect anything in the expected fraction on the OD280 even though the void volume and total volume markers are detected. any ideas or suggetions? thanks very much.
-qkchen-
need more info.
maybe your myoglobin load was too low? can you see the band migrate during the run?
do you have a high enough ionic strength? maybe the myoglobin bound to the sephadex.
what do you use to determine included volume? maybe it elutes too close to the myoglobin so that they are indistinguishable?
-mdfenko-
As mdfenko pointed out, your sample maybe stock on the column. Also, G50 is not the right matrix. you probably need to use G75 at least.
-genehunter-1-
QUOTE (mdfenko @ Feb 19 2008, 02:03 PM)
need more info.
maybe your myoglobin load was too low? can you see the band migrate during the run?
do you have a high enough ionic strength? maybe the myoglobin bound to the sephadex.
what do you use to determine included volume? maybe it elutes too close to the myoglobin so that they are indistinguishable?
maybe your myoglobin load was too low? can you see the band migrate during the run?
do you have a high enough ionic strength? maybe the myoglobin bound to the sephadex.
what do you use to determine included volume? maybe it elutes too close to the myoglobin so that they are indistinguishable?
thanks, most likely there was not enough myoglobin loaded. initially, the distinctive red band could be seen migrating but it dissipated towards the end of the column.
how would one determine a. included volume and b. whether therer is enough ionic strength? thanks.
-qkchen-
QUOTE (genehunter-1 @ Feb 20 2008, 12:27 PM)
As mdfenko pointed out, your sample maybe stock on the column. Also, G50 is not the right matrix. you probably need to use G75 at least.
i was thinking about using G75 also but my supervisor does not like G75. it doesn't have good resolution for resolving smaller peptides at higher fractions, apparently. any feedback would be appreciated.
-qkchen-
QUOTE (qkchen @ Feb 20 2008, 04:05 PM)
thanks, most likely there was not enough myoglobin loaded. initially, the distinctive red band could be seen migrating but it dissipated towards the end of the column.
how would one determine a. included volume and b. whether therer is enough ionic strength? thanks.
how would one determine a. included volume and b. whether therer is enough ionic strength? thanks.
the band always spreads as the sample runs through the column. that's part of the charm of column chromatography. but. i would think that if it was visible to the eye at the start then it should be detectable by uv.
the general rule of thumb for gel filtration is use an ionic strength of 0.2 or higher (that's what they say now, in the past they said 0.1 then 0.15).
to help answer your questions i am attaching a pdf of the gel filtration handbook from ge healthcare (they have a number of handbooks available for free download).
-mdfenko-
QUOTE (mdfenko @ Feb 20 2008, 03:09 PM)
QUOTE (qkchen @ Feb 20 2008, 04:05 PM)
thanks, most likely there was not enough myoglobin loaded. initially, the distinctive red band could be seen migrating but it dissipated towards the end of the column.
how would one determine a. included volume and b. whether therer is enough ionic strength? thanks.
how would one determine a. included volume and b. whether therer is enough ionic strength? thanks.
the band always spreads as the sample runs through the column. that's part of the charm of column chromatography. but. i would think that if it was visible to the eye at the start then it should be detectable by uv.
the general rule of thumb for gel filtration is use an ionic strength of 0.2 or higher (that's what they say now, in the past they said 0.1 then 0.15).
to help answer your questions i am attaching a pdf of the gel filtration handbook from ge healthcare (they have a number of handbooks available for free download).
i used even more myoglobin the second time so i was able to see the red color both in the collected fractions and during the column run throughout the length of the column. thanks for your help.
-qkchen-