Magnetic beads IP Protocol

Protocol (Protein A/G/L)

The protocol (Fig. 1) uses 50 μL Immunomagnetic Beads Protein A, but this can be scaled up or down as required.

Cell Lysis

Cells may be lysed using any standard cell lysis protocol in accordance with your starting materials. We suggest using NP40 Cell Lysis Buffer (supplied with kit).

Prepare Beads

1. Resuspend Immunomagnetic Beads in the vial (vortex >30 sec or tilt and rotate 5 min).
2. Transfer 50 μL Immunomagnetic Beads to a tube.
3. Place the tube on the Magnetic Separator to separate the Immunomagnetic Beads from the solution, and remove the supernatant. 4. Remove the tube from the Magnetic Separator.

Bind Antibody

1. Dilute your antibody (typically 1–10 μg) in 200 μL PBST and add to the Immunomagnetic Beads. The amount of antibody used needs to be optimized.
2. Incubate and rotate for 10 min at room temperature.
3. Place the tube on the Magnetic Separator and remove the supernatant.
4. Remove the tube from the Magnetic Separator and wash the Immunomagnetic Beads using 200 μL PBST, by gentle pipetting. For storage of antibody-conjugated Immunomagnetic Beads, we suggest to use PBST to prevent aggregation.

Immunoprecipitate Target Antigen

1. Place the tube (from Bind Antibody step 5) on the Magnetic Separator and remove the supernatant.
2. Add your sample (typically 100–1,000 μL) to the tube and gently mix the Immunomagnetic Beads-antibody complex by gentle pipetting 3. Incubate with rotation for 10 min at room temperature to allow antigen to bind to the Immunomagnetic Beads-antibody complex.
Note: it may be necessary to increase incubation times for optimal binding.
4. Place the tube on the Magnetic Separator and remove the supernatant or transfer the supernatant to a clean tube for further analysis, if desired.
5. Wash the Immunomagnetic Beads-antibody-antigen complex 3 times using 200 μL PBS for each wash. Separate on the Magnetic Separator between each wash, remove the supernatant and resuspend the complex by gentle pipetting.
6. Resuspend the above complex in 100 μL PBS and transfer the  Immunomagnetic Beads suspension to a clean tube. This is recommended to avoid co-elution of proteins bound to the tube wall.

Elute Target Antigen

A. Denaturing Elution
1. Place the tube (from step 6 in Immunoprecipitation of Target Antigen) on the Magnetic Separator and remove the supernatant.
2. Add 20 μL Elution Buffer and 5 μL 5×SDS-PAGE Sample Buffer and resuspend the Immunomagnetic Beads complex by gently pipetting.
3. Heat for 5-10 min at 95-100 ℃.
4. Place the tube on the Magnetic Separator and collect the supernatant for further analysis.

B. Mild Elution
1. Place the tube (from step 6 in Immunoprecipitation of Target Antigen) on the Magnetic Separator and remove the supernatant.
2. Add 20 μL Elution Buffer and gently pipette to resuspend the complex. Avoid foaming.
3. Incubate with rotation for 2 min at room temperature to dissociate the complex.
4. Place the tube on the Magnetic Separator and transfer the supernatant containing eluted antibody and antigen to a clean tube. If the eluted protein is to be used for functional assays or stored, the pH of the eluate can be adjusted by adding Neutralization Buffer.

Target Antigen Detection

A. Denaturing Elution
1. SDS-PAGE for staining and protein identification
2. SDS-PAGE for Western blotting
3. SDS-PAGE for Fluorography

B. Mild Elution
1. Protein characterization
2. Immunization
3. Enzyme studies
4. AA sequence determination
5. Crystallization
C. No Elution

1. Protein interaction
2. Enzyme studies
3. Bioassays
4. Immunoassays

Protocol(For tagged protein)

The protocol (Fig. 1) uses 50 μL FLAG Tag Immunomagnetic Beads, but this can be scaled up or down as required.
Cell Lysis
Cells may be lysed using any standard cell lysis protocol in accordance with your starting materials. We suggest using NP40 Cell Lysis Buffer (supplied with kit).
Immunoprecipitate Target Antigen
1. Add 50 μL of Immunomagnetic Beads into a 1.5 mL microcentrifuge tube.
2. Add 150 μL of 1× TBST buffer to the Immunomagnetic Beads and gently vortex to mix.
3. Place the tube into a Magnetic Separator to collect the Immunomagnetic Beads against the side wall of the tube. Remove and discard the supernatant.
4. Add 1 mL of 1×TBST buffer to the tube. Invert the tube several times or gently vortex to mix for 1 min. Collect the Immunomagnetic Beads with a Magnetic Separator. Remove and discard the supernatant.
5. Add the sample containing target protein (~100 μg of protein in 100 μL) to the pre-washed Immunomagnetic Beads, add 400 μL of 1×TBST buffer and incubate at room temperature for 30 min with mixing.
6. Collect the Immunomagnetic Beads with a Magnetic Separator , remove the unbounded sample and save for analysis.
7. Add 300 μL of 5× TBST buffer to the tube and gently mix. Collect the Immunomagnetic Beads and discard the supernatant. Repeat this wash twice.
8. Add 300 μL of ddH2O to the tube and gently mix. Collect the Beads on a Magnetic Separator and discard the supernatant.
Elute Target Antigen
A. Neutral Elution Protocol
1. Prepare DYKDDDK peptide (PP101274) at 1mg/mL in PBS
2. Add 50 μL 1mg/mL DYKDDDK peptide to the Immunomagnetic Beads, gently vortex to mix and incubate the sample at 37 oC on a rotator for 5-10 min. Elution may be performed at reduced temperatures, but lower yields may result.
3. Separate the Immunomagnetic beads on a Magnetic Separator and save the supernatant containing the target antigen.
4. Repeat Elution step once for higher recovery.
B. Alkaline Elution Protocol
1. Add 100 μL of Alkaline Elution Buffer to the tube.
2. Gently vortex to mix and incubate the sample at room temperature on a rotator for 5 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the target antigen.
4. To neutralize the sample, add 50 μL of Neutralization Buffer for each 100 μL of eluate.
C. Acidity Elution Protocol
1. Add 100 μL Acidity Elution Buffer.
2. Gently vortex to mix and incubate the sample at room temperature on a rotator for 5-10 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the target antigen.
4. To neutralize the low pH, add 15 μL of Neutralization Buffer for each 100 μL of eluate.
D. Elution Using Sample Buffer
1. Add 100 μL of SDS-PAGE Sample Buffer to the tube.
2. Gently vortex to mix and incubate the sample at 95-100⁰C for 5-10 min.
3. Magnetically separate the Immunomagnetic Beads and save the supernatant containing the antigen.