抗體純化試劑盒：填料為原裝的GE??！國(guó)產(chǎn)的價(jià)格！！
抗體小量純化試劑盒，來源于金黃色葡萄球菌的蛋白A與抗體的不變區(qū)有多個(gè)結(jié)合位點(diǎn)。將蛋白A與Sepharose共價(jià)交聯(lián)制備的層析介質(zhì)，可以用于抗體純化?？贵w與蛋白A在高鹽高pH值條件下結(jié)合，在低鹽pH值條件下解離。不同來源的抗體與蛋白A的結(jié)合能力有較大差異，結(jié)合與解離的條件也有區(qū)別。
訂貨信息：
中文名稱：抗體小量純化試劑盒（10T）
英文名稱：Antibody Purification Kit
產(chǎn)品貨號(hào)：ZKCY-18110
規(guī) 格：10次
價(jià)
格：詢價(jià)
Protein A對(duì)不同來源IgG結(jié)合劑量
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高親和性（如兔）
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低親和性（如小鼠）
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Protein A Sepharose 4 FF結(jié)合劑量
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35 mg/ml
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3-10 mg/ml
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Protein A Sepharose 4 FF每次用量
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40微升
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40微升（50%懸液）
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每次純化抗體數(shù)量
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700 微克
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60-200微克
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一般高滴度抗血清中IgG濃度
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小鼠
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兔抗血清
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羊
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IgG濃度
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10 mg/ml
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5 mg/ml
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20 mg/ml
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Mini kit
PRE-EQUILIBRATION
1. Equilibrate the Spin Column with 0.6 mL Binding Buffer A by centrifuging the spin column at 500 × g for 1 minutes.
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 μm Syringe Filter to remove any debris immediately before loading the sample. Protein precipitation is common during storage and when repeated freeze/thaw cycles in ascites, sera and tissue culture supernatants occur. Newly formed aggregates and precipitating protein complexes can foul the Protein A media and result in significantly slower flow rates. Ensure that the samples are filtered immediately before loading, using filters with pore sizes no greater than 0.22 μm. It is critical to the optimal performance of these devices that these instructions are rigorously followed.
SAMPLE LOADING
3. Dilute the filtered sample 1:1 v/v in Binding Buffer A. (For example, add 1 mL filtered sample to 1 mL buffer.) Pipette the 0.6 mL sample into the spin column. Centrifuge the spin column at 100–150 × g for 1 minutes. Ideal
sample loading conditions are obtained using a flow rate of less than 1 mL/min. It may be necessary to increase the spin time or spin speed if any sample remains in column. Alternatively, a flow rate slower than expected is indicative
of a partially clogged plug resulting from incomplete filtration of the sample.
WASHING
4. Wash the spin column to remove unbound contaminants by adding 0.6 mL Binding Buffer A and centrifuging the spin column for 1 minutes at 500 × g. Add another 0.6 mL Binding Buffer A and centrifuge for 2 more minutes at 500 × g. The unbound wash will contain non-immunoglobulin components. 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 speed to 1000 × g with 2 minute spin time bursts.
ELUTION
■ For purifying mouse IgG1, rat IgG1, rat IgG2a, rat IgG2b and bovine IgG1 (or if you are unsure which IgG subclass you are purifying), use both elution steps 5 and 6 for your initial kit use in order to establish the mildest elution condition possible 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 speed to 1000 × g with 2 minute spin time bursts.
5. Elute the bound IgG with 0.1 mL Elution Buffer B1 directly into a fresh centrifuge tube containing 0.005 mL Neutralization Buffer 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 with 0.1 mL Elution Buffer B2 directly into a fresh centrifuge tube containing 0.013 mL Neutralization Buffer C to bring the sample to neutral pH. Centrifuge the spin column for 5 minutes at 500 × g. Save the sample for analysis.
REGENERATION
7. Wash the media with 0.6 mL Elution Buffer B2 by centrifuging the spin column at 500 × g for 2 minutes.
Re-equilibrate the media with 0.6 mL 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 with 30,000 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.
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