Rapid Antibody Concentration with Amicon Ultra Centrifugal Filters


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Posted by Millipore on April 01, 2009 at 14:29:43:

Ultrafiltration offers a fast and convenient way to concentrate antibodies purified from serum, ascites fluid, or hybridoma supernatants. The traditional purification protocol for immunoglobulins includes affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low pH buffer. Subsequently, purified antibodies are often too dilute for their intended purpose or for long-term storage. In addition, harsh elution conditions sometimes require buffer exchange to preserve protein activity.



Dialysis is often used for antibody concentration and buffer exchange. However, ultrafiltration provides a quick, alternative method to concentrate and diafilter immunoglobulins with up to 99% recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange with Amicon Ultra and Microcon Centrifugal Filters)


To demonstrate the suitability of Amicon Ultra devices for concentrating purified IgG, rabbit serum was processed with Montage PROSEP-A and PROSEP-G Antibody Purification Kits (Millipore) and then concentrated with Amicon Ultra-15 30K NMWL devices. Figure 1.1 shows a decrease in the retentate volume proportional to the increase in antibody concentration. A twenty-minute centrifugation resulted in > 95% recovery of immunoglobulins. Figure 1.2 shows an SDS-PAGE gel of purified rabbit IgGs before and after ultrafiltration.




Figure 1.1 Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were purified using Montage PROSEP-A or PROSEP-G Antibody Purification Kits. The lines show IgG volume reduction, while the bars show a proportional increase in IgG concentration after 20 minutes of centrifugation time.





Figure 1.2 SDS-PAGE gel of purified rabbit IgG before and after concentration with Amicon Ultra-15 devices.




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