Product Use Citations
Customer Product Validation(13)
Co-immunoprecipitation assay was performed to prove the interaction of FUZ and BNIP3. A549 cells were transfected with Flag-FUZ and BNIP3-His plasmids. Flag-RIOK plasmid was used as negative control.
Data from Biomedical Research Foundation of the Academy of Athens
Data from Shanghai Center for Systems Biomedicine
Bimake Anti-Flag Affinity Gel displays an advantage over Sigma ANTI-FLAG M2 Affinity Gel in FLAG-tagged protein binding capacity.
Cell line: Protein-FLAG (85 kD) overexpressed HEK293T
1 1% Input,
2 Bimake Anti-Flag Affinity Gel,
3 Bimake Anti-Flag Affinity Gel + IP,
4 Sigma ANTI-FLAG® M2 Affinity Gel + IP
Data from Lab of Professor Dahua Chen,Institute of Zoology,CAS
Data from Washington University in St. Louis School of Medicine
Red arrow shows the target protein in elution.
TEV is a protease which is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.
Data from National Center for Protein Science Shanghai
Cell line: Protein-FLAG (90 kD) overexpressed HEK293T
1 ANTI-FLAG Affinity Gel + IP (Protein-FLAG (90 kD) overexpressed HEK293T)
2 ANTI-FLAG Affinity Gel + IP (HEK293T)
Cell line: Protein-FLAG (90 kD) overexpressed HEK293T
1 Sigma ANTI-FLAG® M2 Affinity Gel
2 Sigma ANTI-FLAG® M2 Affinity Gel + IP
3 Bimake Anti-Flag Affinity Gel
4 Bimake Anti-Flag Affinity Gel + IP
Cell line: Protein-FLAG (40 kD) overexpressed HEK293T
1 2% Input,
2 Bimake Anti-Flag Affinity Gel,
3 Bimake Anti-Flag Affinity Gel + IP,
4 Sigma ANTI-FLAG® M2 Affinity Gel + IP
a, HEK293T cells were co-transfected with relevant plasmids, cells were collected at 48 h after transfection and immunoprecipitated with anti-Flag gel (Bimake, B23101). “*” non-specific bands. b, HEK293T cells co-transfected with GFP-SUMO1 or its SUMOylation mutant and Flag-α-catenin. Cell extracts were inmmunoprecipitated with Flag gel described in a.
(A) Anti-Flag resin pull down of mEphA4LBD and untagged zRETECD with immobilized zefn-A5ECD. The black arrows in Lanes 3 and 8 point to the mEphA4LBD pulled down by zefn-A5ECD. (B) Protein G bead pull down of zRETECD and zefn-A5ECD with immobilized mEphA4LBD. The black arrows in Lanes 1 and 4 mark the zefn-A5ECD pulled-down by mEphA4. Molecular weight standards: PageRuler Plus standard protein ladder in Lanes 1(A) and 2(B). PD: samples eluted from the beads after pull down. Lanes without the PD label contain input protein samples as indicated.
Price Comparison
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Description
Anti-Flag Affinity Gel is a Protein A purified murine IgG2b Monoclonal antibody covalently attached to sepharose 4B
Properties
| Antibody clone | 1E6 |
| Antibody isotype | Mouse IgG2b |
| Antibody purification | Protein A purified |
| Antibody bound | NLT 7.5 grams antibody/Liter gel |
| Application | Protein Purification, Immunoprecipitation |
| Recommended volume | IP: 5 μl gel for 500 μl crude protein solution |
| Binding capacity | Minimum 1.1 mg of a Flag fusion protein eluted per ml of packed resin |
| Binding characteristics | Met-N-terminal FLAG fusion protein: Met-FLAG–Protein
N-terminal FLAG fusion protein: FLAG–Protein
C-terminal FLAG fusion protein: Protein-FLAG |
| Shipped in | Blue ice |
Storage
The unopened product is stable at -20°C for 2 years. After use, the resin should be cleaned and stored in 50% glycerol with 10mM sodium phosphate, 150 mM sodium chloride, pH 7.4, containing 0.02% sodium azide to protect the product. Do not freeze in the absence of glycerol.
Protocol
This sample protocol is recommended for Immunoprecipitation of DYKDDDDK-tagged (FLAG-tagged) Proteins.
| Affinity gel Preparation | 1. Thoroughly suspend the Anti-Flag Affinity Gel in the vial, for a uniform suspension of the resin. Quickly transfer 10 μl of the gel suspension (about 5μl of packed gel volume) to a fresh tube.
2. Add 0.5 mL TBS. Thoroughly suspend the Anti-Flag Affinity Gel by pipetting. Centrifuge the resin at 5000 rpm for 30 seconds and carefully remove the supernatant. Be sure to remove all of the wash buffer without discarding the resin. Repeat 3-4 times.
3. Add 500 μl of cell periplasmic extracts to the washed resin.
4. Gently agitate samples for 2 hours at 4°C.
5. Centrifuge the resin for 30 seconds at 5000 rpm. Transfer the supernatants to a fresh tube.
6. Wash the resin with 0.5 ml TBS until the OD280 of the supernatant reads <0.05.
7. Extreme pH condition or excess poly Flag peptide can be applied to the resin to elute the Flag-tagged protein. Choose elution methods according to the characteristics of target protein and downstream application.
8. For direct detection of target protein, add 2x SDS-PAGE sample loading buffer at a 1:1 ration to 20 ul gel suspension. Boil the sample for 5 minutes. Centrifuge at 5000 rpm for 30 seconds. Carefully transfer supernatant into a new vial for SDS-PAGE. |
| Sample Binding |
| Resin Washing |
| Elution And Detection |
File Download
Bimake Anti Flag Affinity Gel manual.pdf | MSDS
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