产品资料

Bimake/Anti-Flag Affinity Gel/5 mL Kit/B23102

Customer Reviews
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Description
Storage
Protocol
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Customer Reviews

Product Use Citation(15)

J Cell Mol Med, 2019, 10.1111/jcmm.14322
PubMed: 30993883
Autophagy, 2019, 10.1080/15548627
PubMed: 30957628
Cell Death Dis, 2018, 10(1):1
PubMed: 30578410
Cell Chem Biol, 2018, 25(11):1380-1388
PubMed: 30174312
Int J Biol Sci, 2019, 15(3):544-555
http://www.ijbs.com/v15p0544.htm
Am J Physiol Renal Physiol, 2018, 315(4):F1006-F1018
PubMed: 29897280
Molecules, 2018, 23(6)
PubMed: 29921758
J Neurosci, 2018, 38(33):7248-7254
PubMed: 29718707
Virology, 2018, 522:158-167
PubMed: 30032029
J Virol, 2018, 10.1128/JVI.00426-18
PubMed: 30135121
J Biol Chem, 2018, 293(31):11996-12010
PubMed: 29903906
PLoS One, 2018, 13(6):e0198291
PubMed: 29889908
Oncogenesis, 2018, 7(3):28
PubMed: 29540699
Cell Host Microbe, 2017, 22(1):86-98.e4
PubMed: 28704656
Biochim Biophys Acta, 2017, 10.1016/j.bbagen.2017.03.005
PubMed: 28285006

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 Gel2 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 mEphA4^LBD and untagged zRET^ECD with immobilized zefn-A5^ECD. The black arrows in Lanes 3 and 8 point to the mEphA4^LBD pulled down by zefn-A5^ECD. (B) Protein G bead pull down of zRET^ECD and zefn-A5^ECD with immobilized mEphA4^LBD. The black arrows in Lanes 1 and 4 mark the zefn-A5^ECD 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.

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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.

Bead 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

Bimake provides Poly FLAG Peptide lyophilized powder for protein product elution.

File Download

Bimake Anti Flag Affinity Gel manual.pdf | MSDS

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