PARP1-Trap A User Manual
Contents
1. 1 Elution with Glycine Elution Buffer Resuspend the PARP1 Trap_A beads in 50 100 ul Elution Buffer 200 mM Glycine pH 2 5 by pipetting up and down for 30 sec Make sure that all of the PARP1 Trap_A beads are resuspended Transfer the supernatant to a new tube Then immediately neutralize the solution with 5 10 pl 1M Tris pH 10 4 To increase elution efficiency this step can be repeated Note It is important that the elution step and the neutralization is done at room temperature and that the buffers are also at room temperature Note Use our spin column protocol with spin columns product code sct 10 for easy elution The use of spin columns ensure a minimal loss off the affinity resins during washing 2 Elution with SDS Sample buffer Laemmli Resuspend PARP1 Trap_A beads in 100 pl 2x SDS sample buffer by pipetting up and down Make sure that all of the PARP1 Trap_A beads are resuspended Boil resuspended PARP1 Trap_A beads for 10 min at 95 C to dissociate immune complexes from beads PARP1 Trap_A beads can be collected by centrifugation at 2 500x g for 1 min at room temperature and SDS PAGE is performed with the supernatant 3 Elution with 8 M Urea Resuspend the PARP1 Trap_A beads in 50 100 ul 8 M Urea solution by pipetting up and down Make sure that all beads are resuspended Shake at 700 rpm for 5 min at room temperature Then centrifuge at 2 500x g for 2 min at RT Transfer the supernatant to a new tube To increase2. Limited Use Label License The purchase of this product conveys to the buyer the limited non transferable right to use the purchased amount of the product and components of the product to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly or by implication For information on obtaining additional rights please contact licensing chromotek com PARP1 Trap_A manual 6 Version 2015 10 20
3. elution efficiency this step can be repeated Sample Preparation for Immunoblot Analysis Add 50 ul 2x SDS sample buffer to the collected samples from step 5 Input and step 9 Flow Through Incubate the samples for 10 min at 95 C Spin down the sample before applying to gel PARP1 Trap_A manual 4 Version 2015 10 20 Optional Protocol for immunoprecipitation and elution of proteins from PARP1 Trap_A using Spin Columns Note spin columns product code sct 10 are not included Steps 1 5 describe the preparation of mammalian cell lysate For other types of cells we recommend using 0 5 1 mg of protein extract and start the protocol with Step 6 Harvest mammalian cells Note If you want to make an IP from other cell types like yeast plants etc please use your own protocol for cell lysis and adequate lysis buffer 1 For one immunoprecipitation reaction the use of 10 10 cells approx one 10 cm dish expressing the protein of interest is recommended 2 To harvest adherent cells aspirate growth medium add 1 ml ice cold PBS to cells and scrape cells from dish Transfer cells to a pre cooled tube spin at 500 g for 3 min at 4 C and discard supernatant Wash cell pellet twice with ice cold PBS Add 500 ul ice cold PBS gently resuspend the cells centrifuge at 500 g for 3 min at 4 C Carefully remove supernatant and discard Repeat wash step twice Note The cell pellet can be store for long te
4. 0 mM Tris Cl pH 7 5 150 mM Naci Eanivcie aimannaiareel 0 5 mM EDTA 0 1 SDS 1 Triton X 100 y 1 Deoxycholate a 10 mM Tris Cl pH 7 5 150 mM NaCl Wash Dilution buffer 0 5 mM EDTA Elution Buffer 200 mM Glycine pH 2 5 Elution Buffer alternative 8 M Urea Neutralization Buffer 1 M Tris pH 10 4 120 mM Tris Cl pH 6 8 20 Glycerol 4 i ee buffer SDS 0 04 Bromophenol blue Laemmli 10 B Mercaptoethanol PARP1 Trap_A manual 1 Version 2015 10 20 Related products Support PARP1 Toolbox Code PARP 1 binding protein xt 250 PARP1 Trap_A Kit xtak 20 Binding control agarose beads bab 20 Spin columns sct 10 sct 20 sct 50 Please refer to our support chromotek com FAQ section at www chromotek com or contact We are also happy to hear your feedback and to include your protocol for the cell lysis in our manual PARP1 Trap_A manual Version 2015 10 20 Protocol for immunoprecipitation and elution of proteins from PARP1 Trap_A Steps 1 5 describe the preparation of mammalian cell lysate For other types of cells we recommend using 0 5 1 mg of protein extract and start the protocol with Step 6 Harvest mammalian cells Note If you want to make an IP from other cell types like yeast plants etc please use your own protocol for cell lysis and equivalent lysis buffer 1 For one immunoprecipitation reaction the use of 10 107 cells approx one 10 cm dish expressing the
5. chromo PARP1 Trap_A for Immunoprecipitation of PARP1 Fusion Proteins from cell extract Only for research applications not for diagnostic or therapeutic use Introduction Poly ADP ribose polymerase 1 PARP1 is one of the most abundant proteins in the nucleus and is involved in many cellular processes like DNA repair transcriptional regulation and modulation of chromatin structure PARP1 Trap is excellent for fast and efficient one step isolation of PARP1 and its interacting factors from cellular extract Isolated PARP1 protein may be used further for immunoblot analysis mass spectrometry and enzyme assays PARP1 Trap utilizes small recombinant antibody fragments covalently coupled to the surface of agarose beads Specificity PARP1 Trap binds to PARP1 and not to other members of the PARP family Content Reagent Code Quantity PARP1 Trap_A xta 20 20 reactions 0 5 ml slurry PARP1 Trap_A xta 100 100 reactions 2 5 ml slurry PARP1 Trap_A xta 200 200 reactions 5 ml slurry Beads Bead size 90 um cross linked 4 agarose beads Properties Storage buffer 20 EtOH Stability and Shipped at ambient temperature Upon receipt store at 4 C Storage Stable for 1 year Do not freeze Required Suggested buffer composition solutions Buffer Buffer Composition Lysis buffer CoIP 10 mM Tris Cl pH 7 5 150 mM NaCl For lysis of mammalian cells 0 5 mM EDTA 0 5 NP 40 RIPA buffer 1
6. e The slurry is more efficiently drawn into a wide bore pipette tip We suggest clipping a little off the end of a regular tip to mimic the benefit of a wide bore tip It is important to thoroughly resuspend the Nano Trap beads slurry by vortexing 7 Centrifuge at 2 500x g for 2 min at 4 C Discard supernatant and repeat wash step twice Carefully remove the supernatant with a hollow needle G27 or a small pipetting tip so that the beads pellet is not sucked up Bind proteins 8 Add diluted supernatant from step 5 to equilibrated PARP1 Trap_A beads from step 7 Tumble end over end for 30 min at 4 C 9 Centrifuge at 2 500x g for 2 min at 4 C If required save 50 ul supernatant for immunoblot analysis Flow Through Carefully remove the supernatant with a hollow needle G27 or a small pipetting tip so that the beads pellet is not sucked up Discard remaining supernatant with unbound fractions The beads with the bound proteins are in the pellet Wash beads 10 Resuspend PARP1 Trap_A beads in 500 ul ice cold wash dilution buffer Centrifuge at 2 500x g for 2 min at 4 C Discard supernatant and repeat wash step twice Within the third washing step transfer the diluted beads to a new tube Optional Increase salt concentration in the second washing step up to 500 mM PARP1 Trap_A manual 3 Version 2015 10 20 Elute proteins Depending on your downstream application different elution methods are possible
7. ed Note It is important that the elution step and the neutralization is done at room temperature and that the buffers are also at room temperature 2 Elution with SDS Sample buffer Laemmli Resuspend PARP1 Trap_A beads in 100 pl wash dilution buffer Then transfer diluted beads to a new tube Centrifuge at 1000x g for 30 60 sec to collect beads and remove was dilution buffer supernatant Add 100 pl 2x SDS sample buffer by pipetting up and down Make sure that all of the PARP1 Trap_A beads are resuspended Boil resuspended PARP1 Trap_A beads for 10 min at 95 C to dissociate immune complexes from beads Beads can be collected by centrifugation at 2 500x g for 1 min at room temperature and SDS PAGE is performed with the supernatant 3 Elution with 8 M Urea Resuspend the beads in 50 100 ul 8 M Urea solution by pipetting up and down Make sure that all PARP 1 Trap_A beads are resuspended Close screw cap on top Shake at 700 rpm for 5 min at room temperature Remove bottom plug of the spin column and centrifuge at 1000x g for 30 60 sec To increase elution efficiency this step can be repeated Sample Preparation for Add 50 ul 2x SDS sample buffer to the collected samples from step 5 Input and step 11 Flow Through Incubate the samples for 10 min at 95 C Spin down the sample before applying to gel Immunoblot Analysis Support Please refer to our FAQ section at www chromotek com or contact support chromotek com
8. protein of interest is recommended 2 To harvest adherent cells aspirate growth medium add 1 ml ice cold PBS to cells and scrape cells from dish Transfer cells to a pre cooled tube spin at 500 g for 3 min at 4 C and discard supernatant Wash cell pellet twice with ice cold PBS Add 500 ul ice cold PBS gently resuspend the cells centrifuge at 500 g for 3 min at 4 C Carefully remove supernatant and discard Repeat wash step twice Note The cell pellet can be stored long term at 80 TC Lyse cells 3 Resuspend the washed cell pellet in 200 pl ice cold lysis buffer by pipetting Supplement lysis buffer with protease inhibitors and DNase not included 4 Place the tube on ice for 30 min and extensively pipette the suspension every 10 min 5 Centrifuge lysated cells at 20 000x g for 10 min at 4 C Transfer supernatant lysate to a pre cooled tube and add 300 ul wash dilution buffer to supernatant lysate Discard pellet with cell debris If required save 50 ul of the diluted lysate for immunoblot analysis Input Optional Add protease inhibitors not included to wash dilution buffer Note At this point cell lysate can be stored long term at 80 Equilibrate beads 6 Beads can be equilibrated during incubation step of lysis procedure Vortex PARP1 Trap_A beads intensively and directly pipette 25 yl bead slurry into a new tube with 500 ul ice cold wash dilution buffer and pipette up and down a few times Not
9. rm at 80 C Lyse cells 3 Resuspend cell pellet in 200 pl ice cold lysis buffer by pipetting Supplement lysis buffer with protease inhibitors and DNase not included 4 Place the tube on ice for 30 min and extensively pipette the suspension every 10 min 5 Centrifuge cell lysate at 20 000x g for 10 min at 4 C Transfer supernatant lysate to a pre cooled tube and add 300 ul wash dilution buffer Discard pellet with cell debris If required save 50 ul of the diluted lysate for immunoblot analysis Input Optional Add protease inhibitors not included to dilution buffer Note At this point cell lysate can be stored for long term at 80 C Equilibrate beads 6 Beads can be equilibrated during incubation step of lysis procedure Remove the upper screw cap of a new spin column and snap of the tip from the bottom Keep cap and bottom plug Place the spin column in a 2 ml tube Pipette 500 pl ice cold wash dilution buffer in the spin column 7 Vortex PARP1 Trap_A beads intensively and directly pipette 25 ul bead slurry into the wash dilution buffer in the spin column Pipette up and down a few times Note The slurry is more efficiently drawn into a wide bore pipette tip We suggest clipping a little off the end of a regular tip to mimic the benefit of a wide bore tip It is important to thoroughly resuspend the Nano Trap beads slurry by vortexing 8 Centrifuge at 100x g for 5 10 sec Discard flow through and repeat
10. wash step twice The beads remain on top of the membrane 9 Close column with the bottom plug Bind Protein 10 Add diluted lysate from step 5 to equilibrated PARP1 Trap_A beads from step 8 Screw on upper cap Tumble end over end for 30 min at 4 C 11 Remove the bottom plug from the spin column and loose top cap Place column in a new 2 ml tube Centrifuge at 100x g for 5 10 sec If required save 50 ul flow through for immunoblot analysis Flow Through Discard remaining flow through PARP1 Trap_A manual 5 Version 2015 10 20 Wash Beads 12 Add 500 ul ice cold wash dilution buffer on top of the membrane to resuspend the PARP1 Trap_A beads Centrifuge at 100x g for 5 10 sec Discard flow through and repeat wash steps twice Optional The salt concentration could be increased in the second washing step up to 500 mM 13 Close column with the bottom plug and place in a new tube Elute Protein Depending on your downstream application different elution methods are possible 1 Elution with Glycine Elution Buffer Add 50 ul Elution buffer to PARP1 Trap_A beads Pipette beads up and down for 30 sec Make sure that all of the PARP1 Trap_A beads are resuspended Close screw cap on top Remove bottom plug of the spin column and pipette 5 yl 1M Tris base pH 10 4 in the 2 ml tube for an immediate neutralization Centrifuge at 1000x g for 30 60 sec To increase elution efficiency this step can be repeat
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