Besides precipitation with ammonium sulfate, what are some ways I can concentrate my protein sample?
You may remove excess solvent and smaller moieties by centrifugation through a microconcentrator. This will concentrate your protein sample.
(1) Choose microconcentrator tube (available from a variety of commercial suppliers) with a protein cutoff smaller than the molecular weight of the protein in the sample. Check our Pierce Protein Concentrators PES.
(2) Add 1 µL of 20% w/v SDS to a dry microconcentrator tube (if sample does not already contain SDS).
(3) Slowly add sample (a few microliters at a time) to membrane until membrane is completely wet. Centrifuge to near (but not complete) dryness.
(4) If intention is to desalt sample or buffer exchange: Add ~50 µL water to microconcentrator and spin until nearly dry. Repeat buffer exchange. Sample will remain on membrane. Check our Zeba desalting proteins.
(5) Using a new collection tube, invert membrane and spin at low speed (1000 x g) to elute protein from membrane. Add 2X SDS-Sample Buffer containing 10 mM DTT to membrane: vortex, invert and spin. Final volume should be ~20 µL.
Find additional tips, troubleshooting help, and resources within our Protein Dialysis, Desalting, and Concentration Support Center.
What reagents and conditions are Zeba Desalting columns, plates, and cartridges compatible with?
Zeba Desalting columns, plates, and cartridges are compatible with a range of reagents and conditions. They have been confirmed to be compatible with the following:
- pH range of 3-10
- Mild oxidants/reductants
- Chaotropes (stable in 8 M urea and 6 M guanidine HCl)
- Salts
- Alcohols up to 20%
- Organic solvents such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) (we recommend using a step-gradient of increasing concentration of the organic solvent during column equilibration)
Find additional tips, troubleshooting help, and resources within our Protein Dialysis, Desalting, and Concentration Support Center.
Can I use Zeba Desalting plates with a vacuum manifold, and if so, are there any specific considerations or limitations to keep in mind?
Yes, Zeba Desalting plates can be used with a vacuum manifold. Unfortunately, we do not offer a protocol for the same. It is important to note that the pressure settings of the vacuum manifold will need to be optimized for each instrument as the pressure apparatus may differ. Additionally, it has been reported that protein recovery with Zeba Desalting plates using a vacuum manifold may be lower compared to the traditional centrifugation workflow.
Find additional tips, troubleshooting help, and resources within our Protein Dialysis, Desalting, and Concentration Support Center.