1. Recommended Wash Buffer
|
Wash Buffer
|
TBST: 50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20, pH 7.4
|
|
Elution Buffer A
|
0.15 M Glycine, pH 2.5-3.1
|
|
Elution Buffer B
|
2 mg/mL HA peptide, 50 mM Tris, 150 mM NaCl, pH 7.4
|
|
Neutralization Buffer
|
1 M Tris-HCl, pH 8.0
|
2.Preparation of Magnetic Beads MedChemExpress
2.1 Resuspend the Magnetic Beads in the vial (tilt and rotate for 2 minutes or gently pipette for 10 times, do not vortex). Transfer 10 μL of Anti-HA Magnetic Beads suspension into a new tube.
2.2 Add 500 μL of wash buffer to the beads and gently pipette to mix. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step for 2 times.
3. Protein Binding
3.1 Add 500 μL of cell lysate (the sample containing HA-tagged protein) to the washed beads. For Ag binding, incubate for 2 hours at room temperature or overnight at 4°C while gently rotating the tube.
3.2 Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Note: Occasional aggregation of magnetic beads during the binding process doesn't affect experimental results.
4. Washing Add 500 μL of wash buffer to the Magbeads-Ag complex and mix gently. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step for 4 times.
5. Elution & De tection Three elution methods are recommended according to protein characteristics or further usage:
1) Elution with sample buffer for gel electrophoresis and immuoblotting. Add 50 μL of 1× SDS-PAGE loading buffer to each tube and boil for 5 minutes. Cool and place the tube into a magnetic stand to collect the beads and transfer the supernatant to a new tube. Keep the supernatant containing the target antigen for SDS-PAGE analysis.
2) Elution with Elution Buffer A under acidic condition. Add 50 μL of Elution Buffer A to each tube. Incubate with gentle shaking or on a rotator for 10 minutes at room temperature. Place the tube into a magnetic stand to collect the beads and transfer the supernatant to a new tube. Adding 25 μL of Neutralization Buffer for each 50 μL of eluate to neutralize the low pH, which may help preserve bioactivity of target protein.
3) Elution with Elution Buffer B under native condition. Add 3-5 (v/v) volume of Elution Buffer B to each tube. Incubate with gentle shaking or on a rotator for 1 hour at room temperature or 2 hours at 4ºC. Place the tube into a magnetic stand to collect the beads and transfer the supernatant to a new tube. For immediate use, store the eluates at 4°C, or store at -20°C for long term storage.
Precautions:
1 The pH of Anti-HA Magnetic Beads is 6-8.
2 Do not centrifuge, dry or freeze the magnetic beads. Centrifuging, drying or freezing will cause the beads to aggregate and lose binding affinity.
3 For best results, determine optimal conditions for expression of HAtagged fusion fusion protein before attempting immunoprecipitation.
4 To minimize protein degradation, protease inhibitor cocktails (Cat. No.: GK10014) are highly recommended.
5 For the best experimental performance, it is recommended to use the magnetic stand.
6 Do not use cell lysate containing dithiothreitol (DTT). DTT may cause the HA antibody to leach from the beads.
7 This product is for R&D use only, and is not for drug, house hold, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
Troubleshooting：
|
Problem
|
Possible Cause
|
Solution
|
|
High background
|
Nonspecifically binding of protein to the antibody, magnetic beads or EP tubes.
|
Pre-clear lysate to remove nonspecific binding proteins.
|
|
After suspending beads for the final wash, transfer the entire sample to a clear EP tube and then magnetic separation or centrifugation.
|
|
Washing times were not sufficient.
|
Increase the number and time of washes.
|
|
Little or no c-Myc-tagged protein
is detected
|
No or minimal tagged protein was expressed.
|
Verify protein expression by SDS-PAGE or Western blot analysis of the lysate using an c-Myc-tagged positive control as a reference.
|
|
Increase the amount of lysate used for IP.
|
|
Tagged protein degraded.
|
Prepare fresh lysate.
|
|
Use appropriate protease inhibitors (Cat. No.: GK10014).
|
|
Incubation time was inadequate.
|
Prolong the incubation time.
|
|
Interfering substance was contained.
|
Do not use cell lysate containing dithiothreitol (DTT),2-mercaptoethanol, or other reducing agents.
|
|
Excessive detergent concentration may interfere with the
antibody-antigen interaction.
|
|
|
|
|
;)
,-1-7$$$37316672.jpg');)
;)
;)
$$$36806353.jpg');)
;33(7)%EF%BC%9A546-561$$$37156877.jpg');)
;)
;)
;)
%201124-1138.$$$35908550.jpg');)
%20766-780.$$$37100057.jpg');)
%20418-432.$$$36893736.jpg');)
%EF%BC%9A-240-255.$$$35108512.jpg');)
533-545$$$36543525.jpg');)
%201-13.$$$36600053.jpg');)
;)