Product Description
DYKDDDDK-Nanoab-Magnetic
beads CAT：FM***0
Price:
Product number: FNM********0,
FNM******0k Storage conditions:
2℃*8℃ 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. Â 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. Â 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. Â 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. Â Balance/washing solution: *0 mM Tris, 0.*5 M
NaCl, pH7.4 Â Eluent: 0.1 M Glycine-HCl,
pH3.0 Â Competitive elution solution: *0 mM Tris,
0.*5 M NaCl, ******0 ug DYKDDDDK peptide/ml,
pH7.4 Â Neutralization solution: 1 M Tris-HCl,
pH8.0 Â 3. Sample purification The use of the product
is introduced based on two applications: protein purification
process and IP process. Â
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. Â
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. Â 3) Elution of target
protein: Â 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. Â 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: Â Use competitive elution buffer (DYKDDDDK
peptide content **0 ug/ml) with **5 times the volume of magnetic
beads for elution, incubate at **8℃ 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 4℃, and stored at **0℃ for a long
time. Â 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. Â Add 2XSDS-PAGE Loading Buffer equal to the
volume of magnetic beads to each tube and heat at *5℃ 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. Â Note: Anti-DYKDDDDK-Nanoab-magnetic Beads
have limited regeneration effect, so please use with
caution. Â 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). Â
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. Â 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). Â 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. Â 5) Elution of the target
protein: Â 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. Â Add one-tenth of the elution volume of
neutralizing solution to the elution component and adjust the pH
value to 7.**8.0. Â 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. Â B Competitive
elution: Â Use competitive elution solution (DYKDDDDK
peptide content **0 ug/ml) with **5 times the volume of magnetic
beads for elution, incubate at **8℃ 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 4℃ and stored at **0℃
for a long time. Â 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. Â Add 2XSDS-PAGE Loading Buffer equal to the
volume of magnetic beads to each tube and heat at *5℃ 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.
| Country: |
China |
| Model No: |
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| FOB Price: |
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| Product Group : |
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