I am doing a molecular exclusion in FPLC with Sephacryl S-300 16/60 column. The run progresses and I can not see the chromatogram, the sample seems to stay in the loop or does not enter the column, I do not know what happens. At the injection site I inserted a syringe and extracted a sample, as if it were a lot of sample for the loop, the loop is 500 microliters and inject a little more. Why do not I see the peaks?
Hi Lisandra, There is very little information provided by you.
Please recheck your program -if by mistake you run another program. Did you do pump wash and rule out the possibility of air bubble - How much was your buffer in bottle, and how much it is now ? Whatever you did prior to that did you see a sharp peak and did you run gel and confirmed that you have protein, have you checked if your amicon filters are fine, and did you load that concentrated sample on gel and checked if you see band. If everything is fine then your protein is still in the column hanging at the top ( This is the last possibility to think of )
I'd simply load a dye sample, then you can see if it stays in the loop or at which time it is loaded. Blue dextran is commonly used in SEC, it also marks the excluded volume of the column. No proteins can leave the column before the dextran.
In addition to the other suggestions you can also do a run with markers (gel filtration standard protein mixture) to see if your column still behaves well.
If you are sure your sample contains (your) protein, it also may have precipitated - make sure your protein is 'happy' in the buffer you use for gel filtration.
Dear Lisandra
is not easy to reply to your question. just 3 doubts that can help you to find a solution:
1) in which position was the valve when did you loaded the sample from the sirynge into the loop. ? load or inject? because a common mistake is perform this step in inject and not in load because it sound ad injection..but it refer to the injection from the loop into the coloumn, if you push your sirynge piston to load the sample in this position..your sample will be directed on the waste.
2) At the end of the sample load did
you left the syringe inserted in the injection port or di you remove it? Because you have to mantain it inserted to avoid air bubbles that can cause sample disharge from loop and air injection into the coloumn.
see attached guidelines.
3) less probable: are you monitoring protein at 280nm ? Do your protein contains aromatic aa as w or y? because some proteins are poor of this aa and difficult to be detectable at 280nm.
ciao
Manuele
Thank U very much to all of U for the suggestions. It were very useful!!!!!!!!!
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