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pockypoke 12-26-2012 06:24 PM

Problems with antibody precipitating at concentration
 
Hi!

I am trying to concentrate purified mAb using Amicon (30kDa), but I have major problems with the mAb precipitating during concentration. Is there anything I can do to reduce precipitation?

Thanks in advance!

Zagami 02-28-2013 10:43 AM

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 and activated users can see links. Click Here To Register...]). 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 and activated users can see links. Click Here To Register...]).
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.


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