Antibody Purification Mini Kit
Protein A Sepharose 4 FF結合劑量
Protein A Sepharose 4 FF每次用量
1.Equilibrate the Spin Column with0.6mL Binding Buffer A by centrifuging the spin column at 500 × g for1minutes.
NOTE: If using one spin column, ensure that the spin column is counterbalanced with a unit of equal weight (adjusted
with distilled water).
CLARIFICATION OF SAMPLE
2.Pre-filter the sample (e.g., tissue culture supernatant, serum or ascites) through a 0.22 μmSyringeFilter toremoveany debris immediately before loading the sample.Protein precipitation is common during storage and when repeatedfreeze/thaw cycles in ascites, sera and tissue culture supernatants occur. Newly formed aggregates and precipitatingprotein complexes can foul theProteinA media and result in significantly slower flow rates. Ensure that thesamples are filteredimmediatelybefore loading, using filters with pore sizes no greater than 0.22 μm. It is critical tothe optimal performance of these devices that these instructions are rigorously followed.
3.Dilute the filtered sample 1:1 v/v in Binding Buffer A. (For example, add 1 mL filtered sample to 1 mL buffer.) Pipettethe0.6mL sample into the spin column. Centrifuge the spin column at 100–150 × g for1minutes.Ideal
sample loadingconditions are obtained using a flow rate of less than 1 mL/min. It may be necessary to increase the spin time or spinspeed if any sample remainsincolumn. Alternatively, a flow rate slower than expected is indicative
of a partiallyclogged plug resulting from incomplete filtration of the sample.
4.Wash the spin column to remove unbound contaminants by adding0.6mL Binding Buffer A and centrifuging the spincolumn for1minutes at 500 × g. Add another0.6mL Binding Buffer A and centrifuge for2more minutes at 500 × g.Theunbound wash will contain non-immunoglobulin components. A flow rate slower than expected is indicative of apartially clogged plug resulting from incomplete filtration of the sample. In the unlikely event that flow rates are
significantly slower than those expected, increase the centrifugal speed to 1000 × g with2minute spin time bursts.
■For purifying mouse IgG1, rat IgG1, rat IgG2a, rat IgG2b and bovine IgG1 (or if you are unsure which IgG subclass youare purifying), use both elution steps 5 and 6 for your initial kit use in order to establish the mildest elution conditionpossible for the antibody. Analyze the two fractions in separate tubes to avoid sample dilution.
■For purifying mouse IgG2a, mouse IgG2b, mouse IgG3, rat IgG2c, human IgG1-IgG4, rabbit IgG, guinea pig IgG1,
guinea pig IgG2, bovine IgG2 and any other IgGs, proceed to elution step 6 only.
■A flow rate slower than expected is indicative of a partially clogged plug resulting from incomplete filtration of the
sample. In the unlikely event that flow rates are significantly slower than those expected, increase the centrifugal speedto 1000 × g with2minute spin time bursts.
5.Elute the bound IgG with0.1mL Elution Buffer B1 directly into a fresh centrifuge tube containing 0.005 mL NeutralizationBuffer C to bring the sample to neutral pH. Centrifuge the spin column for 5 minutes at 500 × g.Save the sample for analysis
6.Elute the bound IgG with0.1mL Elution Buffer B2 directly into a fresh centrifuge tube containing0.013 mL NeutralizationBuffer C to bring the sample to neutral pH. Centrifuge the spin column for 5 minutes at 500 × g.Save the sample for analysis.
7.Wash the media with0.6mL Elution Buffer B2 by centrifuging the spin column at 500 × g for2minutes.
Re-equilibrate themedia with0.6mL of Binding Buffer A by centrifuging the spin column at 500 × g for 2 minutes.
DESALTING AND CONCENTRATING
8.If necessary, desalt and concentrate the antibody preparation using the Ultra centrifugal filter device with30,000 NMWL. Add 0.05-0.5% w/v sodium azide if the antibodies are to be stored at 2–8 °C.
We recommend freezing the antibodies in small aliquots in 50% glycerol at -20 °C for long term storage.