Rapid Capturem Protein A-based antibody purification (Phase-out notice: These products will not be available after current inventory is exhausted)

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Capturem Protein A mini spin columns were tested for the ability to purify antibodies from various animal sera: sheep, goat, rat, mouse, human, rabbit, and horse

Capturem Protein A mini spin columns were tested for the ability to purify antibodies from various animal sera: sheep, goat, rat, mouse, human, rabbit, and horse. Columns were first equilibrated with 800 μl of Protein A binding buffer (0.5 M phosphate, pH 8.0 containing 2 M NaCl), and then centrifuged at 1,000 x g for 1 min. Serum samples (250 μl) were diluted in 1 ml of Protein A binding buffer, and 600 μl of sample was loaded on an equilibrated column, followed by centrifugation at at 1,000 x g for 1 min. The loading process was then repeated with another 600-μl sample. The columns were then washed with 800 μl of Protein A binding buffer at 1,000 x g for 1 min. The bound antibody was then eluted with 300 μl of elution buffer (0.1 M glycine, pH 2.5) into a tube containing 30 μl of neutralization buffer (1 M Tris, pH 8.5) to neutralize the eluted antibody. The eluates from various sera were resolved by gel electrophoresis and stained with Coomassie blue. Absorbance at 280 nm was measured with a NanoDrop 2000 spectrophotometer, and used to quantify the amount of antibody in eluted fractions.

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Capturem Protein A Miniprep workflow for antibody purification

Capturem Protein A Miniprep workflow for antibody purification. Each mini spin column can be loaded with up to 800 μl of a diluted sample (antibody sample diluted from 1:1 to 1:20 with buffer). Antibodies are first bound to the membrane, followed by washing with 300 μl of wash buffer, and elution with 300 μl of elution buffer. Over 80% of the antibody can be eluted with as little as 100 μl of elution buffer. Each step is followed by spinning the tube for 1 min at 1,000 x g. The working bed volume of the membrane is <2 μl. This entire purification is complete in ~5 min

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Capturem Protein A miniprep and 96-well plate

Capturem Protein A miniprep and 96-well plate. Capturem Protein A miniprep spin columns accommodate up to 800 μl of antibody sample, and have a capacity of up to 300 μg, depending upon the antibody isotype. Capturem 96-well plates accommodate up to 800 μl of antibody sample per well, with a capacity of up to 300 μg immunoglobulin per well.

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Capturem Protein A mini spin columns were compared against a Protein A resin

Capturem Protein A mini spin columns were compared against a Protein A resin. Sera from the following species were tested: mouse, sheep, goat, rat, rabbit, horse, and human. For the resin (Thermo Fisher Scientific), 20 μl of slurry was used for each purification, following the vendor’s protocol for antibody purification. Storage buffer was first removed, followed by equilibration of the beads with Protein A binding buffer (Thermo Fisher Scientific; 600 μl x 2). The equilibration buffer was then removed and the beads were incubated with diluted serum samples (1 ml of serum in 3 ml of buffer, 1.2 ml of diluted sample) for 4 min with end-over-end rotation. Following loading, the resin was washed twice with 600 μl of Protein A binding buffer, drained, and eluted with 600 μl of elution buffer plus 60 μl of neutralization buffer in a collection tube. Purification steps for the Capturem samples were performed as described in the Capturem Protein A Miniprep Protocol-At-A-Glance. Serum samples were the same concentration and dilution as those used for the Protein A resins. Purities of the final elution samples from both sets were analyzed by gel electrophoresis and stained with Coomassie blue. The amount of antibody eluted was determined by measuring absorbance at 280 nm.

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Capturem Protein A- or G-functionalized membranes

Antibody purification workflow for Capturem Protein A or G 96-well plates. Each well can be loaded with up to 850 µl of a diluted sample (antibody sample diluted from 1:1 to 1:20 with buffer). Following binding, the membrane is then washed and the bound antibiody eluted with the appropriate buffers. Note: Protocol time varies for each format. Check user manuals for specifics on protocol times.

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Capturem spin columns and filtration devices

Available Capturem spin column and filtration device formats. Pictured from left to right: nanoprep, miniprep, 96-well, 24-well, maxiprep, and large-volume Capturem formats. Formats available vary for the different functionalized membranes. Check product pages for list of available formats.

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Load volumes and approximate yields for available Capturem formats by chemistry

Load volumes and yields of Capturem purification formats.

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635717: Capturem Protein A Miniprep Columns

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Capturem Protein A- or G-functionalized membranes

Antibody purification workflow for Capturem Protein A or G 96-well plates. Each well can be loaded with up to 850 µl of a diluted sample (antibody sample diluted from 1:1 to 1:20 with buffer). Following binding, the membrane is then washed and the bound antibiody eluted with the appropriate buffers. Note: Protocol time varies for each format. Check user manuals for specifics on protocol times.

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Capturem spin columns and filtration devices

Available Capturem spin column and filtration device formats. Pictured from left to right: nanoprep, miniprep, 96-well, 24-well, maxiprep, and large-volume Capturem formats. Formats available vary for the different functionalized membranes. Check product pages for list of available formats.

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Load volumes and approximate yields for available Capturem formats by chemistry

Load volumes and yields of Capturem purification formats.

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635720: Capturem Protein A Maxiprep Columns

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635746: Capturem Protein A Buffer