I am looking for a reliable and fast method for separation of protein-fluorescent probe conjugates from the respective free fluorophores present in labeling reactions. HiTrap desalting columns seem to be very fast if used in combination with an AKTA system, but I have never used them before for this purpose. They are smaller than the sizes usually recommended on labeling protocols, so I am a little bit worried about possible contamination of the conjugates with free probes. Besides, I don't know if it is common that fluorescent dyes interact strongly with this particular matrix.
The dyes I am thinking on using are common ones for confocal microscopy, like Alexa Fluor 488, Atto 633, Cy5 etc.
Thank you in advance.
I don't know about your particular system, but for example the company Nanotemper that offers products for MicroScale Thermophoresis have a whole kit for fluorescent labeling of target proteins. and within this kit, there are small gravitational SEC columns for the separation of unlabeled dye and labelled protein. you may look into that to see if it is helpful for you.
What total volume of antibody do you intend to use? Usually we label with similar dyes around 200 µg of antibody in 140 µl batchs and use the PD SpinTrap™ G-25 Desalting Columns (GE, 28918004) to clean up. The SpinTrap allows for a low sample volume (140 µl) to prevent antibody dilution. We also did use HiTrap Desalting columns to clean up Dinitrophenyl-BSA conjugates but with larger volumes.
Kind regards
Martin
You can perform a size exclusion chromatography to remove your fluorophore from protein-fluorophore conjugate. It is one of the efficient techniques for the above purpose. If your protein carries an affinity tag, then you can even perform an affinity chromatography followed by dialysis.
According the the manufacturer, the sample volume for desalting should be no more than 30% of the column volume.
If the labeling reagent concentration is too high, a small portion of it may carry over onto the protein sample. You can reduce this by not collecting the tail end of the protein peak. If it is important to eliminate every trace of labeling reagent, then you can reconcentrate the protein after collecting it (using a centrifugal ultrafiltration unit) and run it through another desalting column to remove the remaining labeling reagent.
You can also put 2 HiTRap columns together in series to increase the capacity.
Dear all,
Thanks for your answers so far.
Martin Paul, my proteins are not antibodies, but enzymes around 50 kDa. I haven't decided the volume yet, but I'm thinking on using the label manufacturer recommendation (usually 1 ml of 1-5 g/L of protein plus the thiol-reactive probe, which I don't expect to be more than a few dozens of microliters). But thanks for the SpinTrap recommendation. It is actually good to know that these spin columns are good enough for the task, as I was a little bit concerned about using them.
Adam B Shapiro, the reconcentration followed by a second purification using the column sounds like a good idea, actually. I may try that out!
Best regards,
Gustavo
Have a look at the PD series of gravity desalting columns from Cytiva (GE). They are available in different sizes for different volumes. PD Midi should work for you if volume is up to 1 ml. There are larger and smaller versions available if you decide on other volumes. They can be used in gravity mode or in spin mode with adapters to fit them into 50 ml centrifuge tubes. I believe a couple of adapters are included in each pack.
Hi,
Just want you to be aware of the available BabyBio Dsalt columns that you can use like the HiTrap columns either with a syringe, pump on the bench or in an ÄKTA system. Comparable with HiTrap, but cheaper.
Typical sample volumes:
BabyBio Dsalt 1 ml: 20 - 300 μl (only available 1 ml prepacked desalting column on the market)
BabyBio Dsalt 5 ml: 100 - 1500 μl
Best regards, Anna
Just keep in mind to use as little SEC resin as possible, as you'll experience losses due to unspecific binding. 0.5mg protein per ml Sephadex is my personal lower limit. If possible, block your column (e.g. with BSA, blocking buffer in your later assay, PBST, etc.). If separation is unsatisfactory, consider increasing salt concentration (e.g. making up to 2M NaCl). This may enhance the retention of some unbound fluorochromes.
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