My lab has a centricon with a minimum of 3 kda cut of . I would like to concentrate a 1 kda peptide. Any suggestions?
The lyophilization method (speed vac or rotovac) is best because of least loss. TCA will not work with 1 kDa protein unless you add a carrier (BSA or glycogen) but still the efficiency is typically 20 to 40%. Depending of what the peptide is dissolved in (and the concentration) the binding to a C18 column may or may not work. Good luck!
Hi!
It depends on how did you previously purify it.
In what buffer is it now?
One solution would be to get it in water (with dialysis), freeze dry it and then resuspend it at the desired concentration with the proper buffer. I not sure if it can help, because you would need anyway a dialysis membrane 100-500 Da and few days of protocol. Moreover you should be careful that your peptide does not precipitate during dialysis. When you freeze dry you could consider to aliquot it before (depending on how much you have now and how much you are going to use in further experiments).
Alternatively you could perform buffer exchange with HPLC using a SEC column and elution with water (maybe it will take you less time).
I hope I was helpful,
Good luck with your experiment,
Cheers,
Diego
you can lyophylize the sample directly provided there is no salt ( buffer salt) or salt present does not interfere with downstream process. if salts are the problem you can purify the peptide by HPLC and then lyophylize
Hi a simple method is degassing. Flow gently a N2 flux into the sample (without touching the solution) and let the sample dry slowly. Good luck!
Freeze drying would be a good way to go with this but you may end up with a high concentration of salts. You could try binding to a reverse phase (hydrophobic) resin and then elute off with acetonitrile/H2O. After releasing you could freeze-dry or blow-dry as suggested by Matteo.
If you don't have an HPLC and columns for reverse phase chromatography, you can simply use Solid Phase Extraction spin columns and elute with a smaller volume of ddH2O.
http://www.thermoscientific.com/en/product/hypersep-c18-spe-cartridges.html
In addition to Arshad's answer, there are 3kd cut off limit filters available through GE healthcare and Amicon. I have worked with higher cut off filters and they work quite well. you might want to give it a try. I managed to concentrate my protein 30 fold. But it might help to do buffer exchange first as you are collecting the filterate.
Try using peptide gel-filtration column (from GE). You can use 30% isopropanol to purify the peptide and you could lyophilize the purified sample to remove the isopropanol. This is just alternative to the HPLC.
Good luck
use Zip-TIPs to concentrate your sample. I used it for concentrating peptides. Its like a pipette tip with C18 stationary phase bound to it. Hope it helps.
If you have dialysis bags with a lower MWCO you can put your protein inside that, and then add PEG 10.000 to the outside. The PEG will pull out the solvent and leave the protein inside concentrated. Again, you need to make sure the protein will not get through the dialysis tubing.
Why don't you just lyophilize it. you can remove it from some ion exchange column with volatile salts like ammonium biocarbonate, and then just rotovap it down.
The lyophilization method (speed vac or rotovac) is best because of least loss. TCA will not work with 1 kDa protein unless you add a carrier (BSA or glycogen) but still the efficiency is typically 20 to 40%. Depending of what the peptide is dissolved in (and the concentration) the binding to a C18 column may or may not work. Good luck!
you can lyophilize your protein- speed or rotavac .hope this will work
Depends on salts etc. Lyophilising and dissolving in smaller volume is a good way but may give very high salt concentration if it's already in buffer. You could use an ion-exchange column or a desalting column to remove the salt first if necessary.
I second Charandeep. A zip tip or, for larger amounts, Sep-Pak cartridge should do it.
why u don't use brad ford method??its very easy and u just need comasii blue powder.
I also would recommend the lyophilization, however it has its own difficulties. Another way would be the use of Millipore centrifugal filters. It's easy and straightforward, though the availability of such cutoff should be checked. At last you can use Mini Dialysis Kit (http://lifescience.scientificlabs.co.uk/product/80648375) if you have no economic problems because of its relatively high price.
If you lyophilize, remember to remove salts first and be sure that the solvent is volatile. otherwise you will concentrate the salts as well. An alternative to lyophilization, if the peptide can withstand heat, is to use a Speedvac centrifugal concentrator. Like lyophilization, depends on vacuum to drive evaporation of the solvent, so you want to remove salts first. Depending upon the volume, you can remove salts by dialysis against water or buffer such as ammonium bicarbonate with volatile components, Or, you can use as suggested reverse phase which comes in many forms. For smaller volumes, you could use a C18 spin column.
Ultrafiltration is a good technique; there are small devices (25 mL) and cut off membranes of 500 and 1000 Da.
Regarding the excellent previous comments, Pall Corporation sells their Microsep Advance Centrifugal Devices which come with a 1000 Da cutoff and can carry up to 5mL solution if you are deciding for filtration as your preferred method.
I agree with Ana Maria Calderon, Ultra-Dia-Filtration is a good technique for concentration. There are small systems with different membranes and cut-off levels for lab use called VIVAFLOW. I would recommend to use cellulose based membranes, which have very low protein binding affinity.
If you have the feeling you re losing some/too much protein by binding to the membrane, you could saturate the membrane with BSA solution prior to using it.
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