Anti flag beads

Manufactured by Smart-Lifesciences

Sourced in China

Anti-Flag beads are a type of laboratory equipment used for protein purification. They are solid-phase affinity beads coated with an anti-Flag antibody, which binds to proteins containing a specific Flag tag sequence. These beads are designed to efficiently capture and isolate Flag-tagged proteins from complex samples.

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Lab products found in correlation

Anti ha beads, by Smart-Lifesciences (1 mentions) Protease inhibitors, by Smart-Lifesciences (1 mentions) Protease inhibitor, by Roche (1 mentions) Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)

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Anti-Flag

4 protocols using anti flag beads

Analyzing Protein Interactions and Modifications in Adipocytes

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The messenger RNA expression level was analyzed by quantitative RT‐PCR. Western blot was used to explore the protein levels. The primer sequences and antibodies used in this study were listed in Table S2 and Table S3, Supporting Information.
For IP assay, HEK293T cell extracts with ectopic expressing tagged plasmids were incubated with anti‐HA beads (Smart lifesciences, SA068001) or anti‐Flag beads (Smart lifesciences, SA042001). Immunoprecipitates were probed with indicated antibodies. To examine the interaction between HDAC1 or ZFP516 with PWWP2B in brown fat cells, lentiviral HA‐PWWP2B or HA‐ZFP516 was transduced to immortalized brown preadipocytes. HA antibody was used to precipitate the HA‐PWWP2B or HA‐ZFP516 protein complex from differentiated mature adipocytes followed by western blot. The HA‐PWW2B protein complex was subjected to LC‐MS/MS analysis (Shanghai Applied Protein Technology Co. Ltd). For the ubiquitination assay, HEK293T cells were treated with 5 µM MG132 for 12 h before harvesting the cells.

Yan L., Jin W., Zhao Q., Cui X., Shi T., Xu Y., Li F., Jin W., Zhang Z., Zhang Z., Tang Q, & Pan D. (2021). PWWP2B Fine‐Tunes Adipose Thermogenesis by Stabilizing HDACs in a NuRD Subcomplex. Advanced Science, 8(16), 2102060.

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Immunoprecipitation of AMPK and SENP2

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HEK-293T cells transfected with HA-AMPKα or FLAG-SENP2 were lysed in lysis buffer containing 50 mM Tris–Hcl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.5% Triton X-100, and protease inhibitors (Roche). After centrifugation, the lysates were incubated with anti-FLAG beads (SMART LIFESCIENCES) for 2 h. The beads were then washed four times with washing buffer (50 mM Tris–Hcl [pH 7.5], 100 mM NaCl, 5% glycerol, 0.1% Triton X-100, and protease inhibitors), and the immunoprecipitates were separated by SDS-PAGE for Western blotting.

Dou X., Zhou W.Y., Ding M., Ma Y.J., Yang Q.Q., Qian S.W., Tang Y., Tang Q.Q, & Liu Y. (2021). The protease SENP2 controls hepatic gluconeogenesis by regulating the SUMOylation of the fuel sensor AMPKα. The Journal of Biological Chemistry, 298(2), 101544.

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Investigating LATS1/2-STAT1 Interactions

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The interactions between LATS1 or LATS2 and STAT1 were investigated using Co-IP assays. Flag-LATS1 or LATS2 and myc-STAT1 were co-expressed in 293T cells for 24 h, and then the cells were lysed in immunoprecipitation buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 0.05mM EDTA, 1% NP40 and 10% glycerol). The cell lysates were immunoprecipitated overnight with anti-FLAG-beads (Smart-Lifesciences). For endogenous Co-IP assays, the KLE cells were lysed in immunoprecipitation buffer and the lysate was pre-cleared with protein-A/G agarose for 2 h at 4 °C. The supernatant was immunoprecipitated overnight with anti-LATS1, LATS2, or STAT1 antibody and subsequently immunoprecipitated with protein-A/G agarose for 2 h at 4 °C. The pellets were washed four times with immunoprecipitation buffer, resuspended in sample buffer, and analyzed by SDS/PAGE (10% gel).

Yang Q., Lv Z., Wang M., Kong M., Zhong C., Gao K, & Wan X. (2024). LATS1/2 loss promote tumor immune evasion in endometrial cancer through downregulating MHC-I expression. Journal of Experimental & Clinical Cancer Research : CR, 43, 54.

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Exogenous and Endogenous Immunoprecipitation Assays

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For the exogenous immunoprecipitation assay, cells were lysed in the 1% NP‐40 buffer (pH = 8.0): 25 mm Tris‐base, 150 mm NaCl, 10% glycerol, and 1% NP‐40; protease inhibitor cocktail (5871, Cell Signaling Technology, Boston, USA) added before use. The lysate (containing 500 µg of total protein) was centrifuged and immunoprecipitated with 20 µL anti‐Flag beads (SA042001, Smart‐Lifesciences, Changzhou, China) at 4 °C overnight. Then, the lysate was centrifuged to remove the supernatant and wash with 1% NP‐40 buffer, followed by Western blot analysis. For the endogenous immunoprecipitation assay, cells were lysed with the IP buffer (pH = 7.5): 50 mm Tris‐base, 150 mm NaCl, 2 mm EGTA, 25 mm NaF, and 25 mm β‐sodium glycerophosphate; protease inhibitor cocktail (5871, Cell Signaling Technology, Boston, USA). The lysate was centrifuged and immunoprecipitated with 20 µL protein A/G Plus‐agarose (sc‐2003, Santa Cruz Biotechnology, Dallas, USA) and a primary antibody (anti‐Bcl‐XL: ET1603‐28, Huabio, Hangzhou, China), IgG antibody (2729S, Cell Signaling Technology, Boston, USA) was used as a negative control. Co‐precipitated protein and initial whole‐cell lysates were analyzed by Western blot.

Yan H., Huang X., Xu J., Zhang Y., Chen J., Xu Z., Li H., Wang Z., Yang X., Yang B., He Q, & Luo P. (2023). Chloroquine Intervenes Nephrotoxicity of Nilotinib through Deubiquitinase USP13‐Mediated Stabilization of Bcl‐XL. Advanced Science, 10(26), 2302002.

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