DYKDDDDK-Nanoab-Magnetic beads By Lablead biotech,

La description

DYKDDDDK-Nanoab-Magnetic beads CATï¼šFM***0 Price: Product number: FNM********0, FNM******0k Storage conditions: 2â„ƒ*8â„ƒ 1. Product introduction The DYKDDDDK tag is a polypeptide fragment composed of eight hydrophilic amino acids, located on the surface of the fusion protein, so it is easier to bind to the corresponding antibody and be decomposed by enterokinase. The DYKDDDDK-Nanoab-magnetic Beads series products have the characteristics of superparamagnetism, rapid magnetic responsiveness, rich hydroxyl functional groups and relatively concentrated particle size, and are important carrier tools in medical and molecular biology research. Anti-DYKDDDDK-Nanoab-magnetic Beads uses anti-DYKDDDDK (DYKDDDDK) nanoantibodies as affinity ligands, which can purify DYKDDDDK tag fusion proteins expressed in prokaryotes, yeast or mammalian cells in one step. 2 Reagent preparation 2.1 Sample preparation Before loading, make sure the sample solution has the appropriate ionic strength and pH value. You can dilute the sample or cell culture medium with the equilibrium solution, or dialyze with the equilibrium solution. It is recommended to centrifuge or filter the sample with a 0.*2 um or 0.*5 um filter membrane before loading to reduce impurities, improve protein purification efficiency and prevent clogging of the column. 2.2 Buffer preparation It is recommended to filter the water and buffer with a 0.*2 um or 0.*5 um filter membrane before use. Balance/washing solution: *0 mM Tris, 0.*5 M NaCl, pH7.4 Eluent: 0.1 M Glycine-HCl, pH3.0 Competitive elution solution: *0 mM Tris, 0.*5 M NaCl, ******0 ug DYKDDDDK peptide/ml, pH7.4 Neutralization solution: 1 M Tris-HCl, pH8.0 3. Sample purification The use of the product is introduced based on two applications: protein purification process and IP process. 3.1 Magnetic bead pretreatment Invert the Anti-DYKDDDDK-Nanoab-magnetic Beads several times to ensure that the magnetic beads are completely mixed, take the calculated amount (calculated according to the sample volume and the sample content) of the magnetic bead suspension, transfer it to a centrifuge tube, place it on a magnetic separator, let it stand for about 1 min, and after the solution becomes clear, use a pipette to discard the clear liquid. Remove the centrifuge tube from the magnetic separator, add the balance solution of the same volume as the suspension, use the gun tip to blow repeatedly 5 times, place the centrifuge tube on the magnetic separator, about 1 min, wait for the solution to become clear, use a pipette to discard the clear solution, and repeat washing 2 times. 3.2 Purification of DYKDDDDK-tagged protein 1) Magnetic bead binding target protein: Add the sample solution to the magnetic bead tube pretreated in step 3.1, vortex and oscillate evenly, place it in a flip mixer at room temperature or gently flip the centrifuge tube manually to promote full contact and adsorption between the sample and the magnetic beads, mix for more than *0 min (the specific time is adjusted according to the binding effect), place it on the magnetic separator, about 1 min, wait for the solution to become clear, absorb and retain the supernatant as a flow-through sample for subsequent detection. 2) Magnetic bead washing: Add 5 times the volume of magnetic bead washing solution to the centrifuge tube, oscillate and suspend, place it on the magnetic separator, about 1 min, wait for the solution to become clear, and discard the supernatant. Repeat this operation more than twice. 3) Elution of target protein: A Acidic elution: Add **5 times the volume of the magnetic beads in the above centrifuge tube (0.1 M Glycine-HCl, pH3.0), pipette 5 times, then place in a flip mixer at room temperature or gently flip the centrifuge tube manually, after ***0 minutes, place on a magnetic separator, about 1 minute, wait for the solution to become clear, aspirate and retain the supernatant, which is the elution component, that is, the target protein. This operation is recommended to be repeated twice. Add one-tenth of the elution volume of neutralizing solution to the elution component and adjust the pH value to 7.**8.0. Note: After acidic elution, the magnetic beads should be immediately balanced with equilibration solution, and Anti-DYKDDDDK-Nanoab-magnetic Beads should not be placed in the elution solution for more than *0 minutes. B. Competitive elution: Use competitive elution buffer (DYKDDDDK peptide content **0 ug/ml) with **5 times the volume of magnetic beads for elution, incubate at **8â„ƒ for *0 min, place the centrifuge tube on the magnetic separator for about 1 min, and carefully remove the supernatant after the solution becomes clear. Do not absorb the filler, which is the elution component. The eluted sample is placed at 4â„ƒ, and stored at **0â„ƒ for a long time. C. Denaturation elution The sample eluted by this method is suitable for SDS-PAGE detection. SDS-PAGE Loading Buffer contains β-mercaptoethanol or DTT, which can disconnect the heavy chain and light chain of the antibody ligand on the filler (size is *0 and *5kDa respectively). In addition, the SDS contained can denature the Ant-DYKDDDDK nanoantibody, and the eluted Ant-DYKDDDDK-Nanoab-magnetic Beads cannot be reused. Add 2XSDS-PAGE Loading Buffer equal to the volume of magnetic beads to each tube and heat at *5â„ƒ for *0 min. Place the centrifuge tube on a magnetic separator for about 1 min. After the solution becomes clear, aspirate the supernatant for SDS-PAGE electrophoresis detection. You can also centrifuge and take the supernatant for electrophoresis detection. Note: Anti-DYKDDDDK-Nanoab-magnetic Beads have limited regeneration effect, so please use with caution. 3.3 IP/Co-IP operation process 1) Magnetic beads bind to target protein: Add the target protein sample containing DYKDDDDK label to the magnetic beads treated in step 3.1 and mix by inversion. Place the centrifuge tube on a mixer and incubate at room temperature for more than *0 min (the specific time is adjusted according to the binding effect). 2) Magnetic bead washing: Place the centrifuge tube on a magnetic separator for about 1 min. After the solution becomes clear, use a pipette to remove the supernatant and retain it for sampling and detection. Add 5 times the volume of the suspension to the centrifuge tube, blow repeatedly ***0 times with the gun tip, place the centrifuge tube on the magnetic separator for about 1 min, wait for the solution to become clear, and discard the supernatant with a pipette. Repeat the above steps 2 times. The target protein-magnetic bead complex is obtained. If an IP experiment is performed, skip steps 3 and 4 and proceed directly to step 5. 3) Binding of target protein and target protein-magnetic bead complex: Add the sample containing the target protein to the treated target protein-magnetic bead complex and mix it upside down. Place the centrifuge tube on a mixer and incubate at room temperature for more than *0 min (the specific time is adjusted according to the binding effect). 4) Magnetic bead washing: Place the centrifuge tube on the magnetic separator for about 1 min, wait for the solution to become clear, remove the supernatant with a pipette, and retain it for sampling and testing. Add 5 times the volume of the suspension to the centrifuge tube, blow repeatedly ***0 times with a pipette, place the centrifuge tube on a magnetic separator for about 1 min, wait for the solution to become clear, and discard the supernatant with a pipette. Repeat the above steps 2 times. The target protein is captured from the mixed system by binding with the target protein. 5) Elution of the target protein: A acid elution: Add **5 times the volume of the magnetic beads to the above centrifuge tube. Acidic elution (0.1M glycine HCl, pH3.0), blow 5 times with a pipette, and then place it in a flip mixer at room temperature or gently flip the centrifuge tube manually. After ***0 minutes, place it on a magnetic separator for about 1 minute. After the solution becomes clear, aspirate and retain the supernatant, which is the elution component, that is, the target antibody. This operation is recommended to be repeated twice. Add one-tenth of the elution volume of neutralizing solution to the elution component and adjust the pH value to 7.**8.0. Note: After acid elution, the magnetic beads should be immediately equilibrated with equilibration solution. Anti-DYKDDDDK-Nanoab-magnetic Beads should not be placed in the elution solution for more than *0 min. B Competitive elution: Use competitive elution solution (DYKDDDDK peptide content **0 ug/ml) with **5 times the volume of magnetic beads for elution, incubate at **8â„ƒ for *0 min, place the centrifuge tube on the magnetic separator for about 1 min, and after the solution becomes clear, carefully remove the supernatant without sucking the filler, which is the elution component. The eluted sample is placed at 4â„ƒ and stored at **0â„ƒ for a long time. C Denaturation elution The sample eluted by this method is suitable for SDS-PAGE detection. SDS-PAGE Loading Buffer contains β-mercaptoethanol or DTT, which can disconnect the heavy chain and light chain of the antibody ligand on the filler (size decibels are *0 and *5 kDa). In addition, the SDS contained in it can denature the Ant-DYKDDDDK nanoantibody, and the eluted Ant-DYKDDDDK-Nanoab-magnetic Beads cannot be reused. Add 2XSDS-PAGE Loading Buffer equal to the volume of magnetic beads to each tube and heat at *5â„ƒ for *0 min. Place the centrifuge tube on the magnetic separator for about 1 min. After the solution becomes clear, take the supernatant for SDS-PAGE electrophoresis detection, or take the supernatant after centrifugation for electrophoresis detection.

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Groupe de produits :	Protein Biology