Antibody ForumAntibody Forum. Ask and discuss antibody suppliers, antibody related techniques and protocols, and antibody production such as using phage display libraries.
Problems with antibody precipitating at concentration
Problems with antibody precipitating at concentration - Antibody Forum
Problems with antibody precipitating at concentration - Antibody Forum. Ask and discuss antibody suppliers, antibody related techniques and protocols, and antibody production such as using phage display libraries.
Re: Problems with antibody precipitating at concentration
Causes of proteins precipitation during Amicon Ultra Centrifugal Filter Devices
1) Over-concentration protein : can lead to precipitation and potential sample loss. Frequently to stop concentration process and mix the concentrate gently using a pipette without introducing air or creating foam, usually every 15 minutes, but this may vary depending on the circumstances.
2) Very high hydrophobicity : entail to formation non-specific binding interactions with the membrane material. For reduction this issue Triton series (e.g. Triton X-100) to use, starting from 0.025 % solution, to inhibit non-specific hydrophobic interactions, and mix it per spin for 15 mins.
3) Isoelectric point : to choose buffer according to this. Never concentrate it in a buffer where the pH equals its isoelectric point, which you can estimate with Expasy protparam software ([Only registered users see links. ]). Also, use a sufficient amount of salt (100 mM NaCl) and for most intracellular proteins, a reducing agent (DTT, β-mercaptoethanol).
4) Not stabilized protein solution (pH, buffers types [TRIS, HEPES, MOPS, MES, PO4] and ionic strength). For to keep solubility and long-term stability can be used 50 mM Arg + 50 mM Glu as buffer additives ([Only registered users see links. ]).
4) Presence of divalent or multivalent cations and urea concentration : preliminary dialysis for decreasing the urea/salts concentration
5) Temperature : influences the precipitation kinetics of proteins, the entropic and enthalpic components of interactions and also the viscosity of water, which is determinant for the filtration speed.
6) Ultrafiltration speed : if the ultrafiltration process is too fast, it could lead to polarization of the concentration of the protein around the membrane, which could lead to unfolding and increase of non-specific protein-protein and protein-membrane interactions.
7) Sample quantity : concentrate only small batches at a time, using temperature and additives as helpers to achieve this.
Check : If your sample turns bright yellowish this may also indicate the formation of soluble aggregates which usually do not disassociate any more. Frequently check your sample during concentration with a photometer. If you see an increase at 320-350 nm this is usually indicative of light-diffraction by aggregated particles. If you see this STOP concentrating. If you use detergents you frequently can usually not use a photometer to determine protein concentrations at 280 nm because many absorb light strongly at this wavelength.