I am using a PD-10 desalting column for a buffer exhange.I need to get an accurate concentration reading on my enzyme, so I am doing a rapid buffer exchange into fresh PBS with 5% glycerol using a PD-10 desalting column. My enzyme is clearly present on a SDS-PAGE gel prior to buffer exchange, and makes a clean spectra with a peak at A280. Then I equilibrated the PD-10 column with 25 mls of PBS, flowed 2 mls of my protein, and eluted in 0.5 ml fractions. I bradford assayed the fractions, and pooled the positive ones. However, the nanodrop spectra on the positive fractions was noise, even using the PBS as a blank. I plan to test the exchanged protein for activity, but this wont help me get an accurate concentration reading. help please
Sounds like some crud in your glycerol. 5% is a lot of stuff.
I would push in into plain PBS first, measure concentration, and then add glycerol later.
The glycerol doesnt affect the nanodrop spectra prior to buffer exchange. and the nanodrop claims to tolerate up to 10% glycerol, but it cant hurt to try. I am also going to try a different buffer and see if the result is the same, and see if the pooled fractions from the exchange exhibit activity
I usually get very clean spectra after PD-10 column. Is your protein prone to aggregation? Maybe it aggregates in PBS which could compromise the spectra.
You can try BSA as a control protein and see if you observe similar results.
I miss in which solution is the protein. And how you elute, PBS? (I mean when 5% glycerol is present)
For such precise experiment I would use a fresh new column.
Hi Bob.
Our lab has used PD-10 columns for buffer exchange and desalting practically forever. With one exception we have never had any issue with downstream assays or noticed leaching of anything out of the column. As mentioned the most obvious cause is contamination. Something similar happened in my lab a long time ago when a student used a box of PD-10 columns that had been expired for several years (!) and had something nasty growing in it.. That produced some hard to explain results in the downstream assays. The other option is protein loss and as a result a very low absorption when you measure absorption.
Gernot
Hi
Are you sure that your protein is within the exclusion limit of the column?
If I remember well, to have these columns working you have to load 2.5 mL of your sample and if your volume is less than this you have to complete up to 2.5 with your equilibration buffer. In addition, which is your elution volume? It should be 3.5 mL. Of course you can take aliquots of 0.5 to test but you have to load the whole volume in one shot.
I hope you find this comments useful. Good luck and let us know how you solved the problem.
Christopher
I'm not exactly sure how old the columns are or how properly they were stored. I tried using a new column from the same box today. I also tried a 50 mM tris/ 150 mM NaCl buffer today, and the results were similar to the PBS. perhaps the columns are contaminated. I also used the tris buffer as a blank, and then flowed some buffer through the column. I nanodropped the flow through, and found that there was some contaminant absorbing mildly in the 230- 260 nm range... I'm going to try with BSA tomorrow to make sure it's not my protein being funny. Also, the eluted protein is still active; which confuses me. Thanks for all the suggestions :)
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