I am doing gel filtration experiments using sephadex g-75 superfine column. I am using 0.1% triton X-100 in 100 mM PBS as elution buffer and getting negative peaks for 46KDa protein. Can any one suggest me why this is happening? As i have never seen negative peaks in gel filtration experiments before.
Hi Chn,
Thanks for your response. I have equilibriated five CV before i loaded my protein into the system.
And to make sure i had repacked the column again with fresh beads swelled at 90 degrees for 3 hours in the same elution buffer.
But the result was the same with negative peaks.
It would be really helpful if you can help me out with this.
Hi,Mathesh
you have done so well!good job! I'm so sorrry to tell you I can not explain the problem you seen.
but there are some reasons about negative peaks in HPLC/FPLC:
negative peak generally is the solvent peak, may be 1: the choice of mobile phase is inappropriate, for example, high UV absorption background, then absorb the solute is lower than the mobile phase background absorption occurs negative peaks. 2: I do not know what you're using chromatography, some instrument settings can also cause problems inverted the peak; 3 samples than the mobile phase low background presence of substances, such as detergent ,inorganic salts, etc., will inverted the peak.
best regards,good luck!
Hi Mathesh,
I think one of the main explanation may be the triton X100 itself : It does absorb a lot in UV with a spectra that looks very similar to a protein spectra. In my recollection, a 0.1% triton solution would give you a UV signal at 280 nm of about 1.8-2 OD. The other parameter is also that your concentration is higher than the critical micellar concentration (0.2mM or 0.013%) and the average micelle size for Triton X100 is about 90 kDa. So maybe you are saturating your detector with the buffer alone, or when your protein is running on the gel filtration it exclude part of the detergent and as the detergent absorbs more, you see a negative peak ? Solutions may be to use much less triton (15-20x less ?), change detergent or to use another denaturation method (urea ?) or detect your protein at a different wavelength (212 nm ?) or with another detection method ?
Good luck,
Nicolas
Yes, Chn and Nicolas are correct regarding high background absorbance by your buffer at the wavelengths you use after auto zero and then having very little dynamic range in the detector. I have had this experience. Sometimes it is due to impure materials used to make your buffers. Sometimes the flow cell needs to be cleaned because of high background or even a weak lamp intensity may reduce your signal and compound your problem with the buffer. What's the path length of the cell and what would happen if you ran your buffer system but monitor in the visible wavelength using a sample that absorbs in that region?
Just one comment regarding column equilibration. as opposed to other types of chromatography, 5 CV may not be enough - depending on the exact composition of the buffer you're using at the moment, compared to the buffer used previously. low salt buffers may take the whole night to equilibrate while high salt buffers may take a couple of hours..
Are the buffer components stable to 90 c for 3 hours (the way you prepared the resin)? How old is the resin? Are there unusually high back pressure changes which can affect detector response? How about checking the eluted column buffer during equilibration against water and against the sample to be applied using a standard spectrophotometer using a 1 cm cuvette at different wavelengths? What happens if you switch to a different vendor's Triton? Could there be any conversion to high UV absorbing solutes from the buffer or resin that bind to/leach out of the resin and cause high background beyond the sample's abosrbance; assuming this is not a refractive index detector and the BUFFER THE SAMPLE IS IN IS EXACTLY THE SAME AS THE EQUILIB BUFFER?
We were taught to initially allow swelling of a gel in a beaker and remove/decant lighter particles repeatedly before loading the gel into a column with a sinter base. Being gel filtration, equilibration and elution/mobile phase r the same. True, detergents are usually employed at 2-3 times the CMC. Equilibration is preferred to be 10 bed volumes. SAMPLE VOLUME SHOULD NEVER EXCEED 5% OF THE VOID VOLUME or a max of 2% bed vol. Detection at 280 nm should first come to a zero OD baseline. Then the chances of a negative peak is not possible. A protein lacking F, Y and W will show no absorbance at 280; they r measured at 225 &
215.
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