[原创]免疫共沉淀（Protein A/G-agarose）试剂盒

macgene 发表于 2009-1-5 09:49:00

mmunoprecipitation Kit   Protein A/G-agarose免疫共沉淀（Protein A/G-agarose）试剂盒Catalogue #: IPK<chmetcnv wst="on" unitname="a" sourcevalue="1" hasspace="False" negative="False" numbertype="1" tcsc="0">001A</chmetcnv>/G   Storage: -20 ºC  Description: The Protein A/G-agarose Purification Kit is designed for rapid purification of proteins expressed in cultured cellsincluding mammalian cells, insect cells, yeast and E. coli. The easy-to-follow procedure is based on novel proteinpurification chemistry. Purification may take place under native conditions or under denaturing conditions dependingon the solubility and/or desired application of the expressed protein. The purified protein can be used directly forenzymatic assays, protein biochemical analyses, SDS-PAGE, as well as other protein based applications. Size:  30 standard assays Kit components: Components Name Cat# SizeComponent A Protein A/G-agarose IR005 1 mLComponent B Binding Buffer N/A 50 mLComponent C Washing Buffer (5X) N/A 50 mLComponent D Elution Buffer N/A 10 mLComponent E Neutralization Buffer N/A 1 mLReagents needed, but not provided in the kit:􀂗
DTT (Cat. #: MC010)􀂗
Phosphate Buffered Saline (PBS) (Cat. #: CC008)􀂗
Proteinase Inhibitor Cocktails (Cat. #: MP027)

macgene 发表于 2009-1-5 09:49:00

B. Preparation of Protein A-agarose/antibody Complex and Immunoprecipitation 1. Thoroughly suspend the protein A/G-agarose beads.2. Transfer 30ul of the gel suspension to a 1.5ml eppendorf tube. (For beads transfer, use plastic pipette tip with theend cut for about <chmetcnv wst="on" unitname="mm" sourcevalue="2" hasspace="False" negative="False" numbertype="1" tcsc="0">2mm</chmetcnv> to allow the beads to be transferred).3. Centrifuge the beads briefly to bring the beads to the bottom of the tube.4. Wash the beads twice with 0.5 ml 1X Washing Buffer.5. Add 0.5 ml Binding Buffer and up to 2 ug antibody against the protein of interest.6. Incubate for 30 minutes with gentle rotating at RT.7. Centrifuge the beads briefly to bring the beads to the bottom of the tube.8. Discharge the supernatant and wash the beads twice with 0.5 ml 1X Washing Buffer.9. Apply 0.5-1 ml of cell lysates (up to 1mg) to the beads. The lysates could be diluted with Binding Buffer.10. Incubate for 2 hours-overnight with gentle rotating at 4ºC.11. Centrifuge the beads briefly to bring the beads to the bottom of the tube.12. Discharge the supernatant and wash the beads >5 times with 0.5 ml 1X Washing Buffer each.  C. Elution Elution with <chmetcnv wst="on" unitname="m" sourcevalue=".1" hasspace="True" negative="False" numbertype="1" tcsc="0">0.1 M</chmetcnv> glycine HCl, pH 2.51. Add up to 300ul Elution Buffer supplemented with <chmetcnv wst="on" unitname="mm" sourcevalue="1" hasspace="True" negative="False" numbertype="1" tcsc="0">1 mM</chmetcnv> DTT to each sample.2. Incubate the samples and controls with gentle shaking for 10 minutes at room temperature.3. Centrifuge the beads for 30 seconds at 5,000 x g. Transfer the supernatants to a new tube containingNeutralization Buffer (1/10 volume of Elution Buffer).Note: The procedure should be performed at room temperature. Do not leave the beads in this buffer >20 minutes.Elution with SDS-PAGE Sample Loading Buffer1. Add 30ul of 2X sample loading buffer (Cat. #: MP006.1) to each sample.2. Boil the samples for 5 minutes.3. Briefly votex the tube and centrifuge the samples at 5,000 x g for 30 seconds to pellet agarose. Transfer thesupernatants to a new tube.4. The samples are ready for loading on SDS-PAGE and immunoblotting using Anti-FLAG or specific antibodiesagainst the fusion protein or associated proteins.Note: The procedure should be preformed at room temperature. Sample buffer should be at room temperature beforeuse.

macgene 发表于 2009-1-5 09:50:00

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macgene 发表于 2009-3-30 14:11:18

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详情请登录网站：www.macgene.com，联系电话：010-82057786

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[原创]免疫共沉淀（Protein A/G-agarose）试剂盒