Ripa extraction reagent

Manufactured by Meilun

Sourced in China

RIPA extraction reagent is a solution used for the extraction and solubilization of proteins from cells and tissues. It is a commonly used buffer in molecular biology and biochemistry applications.

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Lab products found in correlation

Protease inhibitor, by Meilun (3 mentions) Protease inhibitor, by Beyotime (1 mentions) 0.45 μm pvdf membrane, by Merck Group (1 mentions) Usp37, by Proteintech (1 mentions) Bca kit, by Beyotime (1 mentions) Gapdh, by Proteintech (1 mentions) Protein a g plus agarose beads, by Beyotime (1 mentions)

5 protocols using ripa extraction reagent

Co-IP Assay of Protein Interactions

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Cells were harvested at 4°C using RIPA extraction reagent (Meilun) containing protease inhibitors (Meilun) after washed three times with prechilled PBS. Co‐IP assay was performed as we previously described.
⁷⁶ (link) Each experiment was repeated in triplicate at least three times.

Tang J., Long G., Xiao D., Liu S., Xiao L., Zhou L, & Tao Y. (2023). ATR‐dependent ubiquitin‐specific protease 20 phosphorylation confers oxaliplatin and ferroptosis resistance. MedComm, 4(6), e463.

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Western Blot Analysis of Protein Targets

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Cell pellets were harvested and then lysed using RIPA extraction reagent (Meilun) supplemented with protease inhibitors (Beyotime). The BCA kit (Beyotime) was applied to detect the protein concentration. Afterward, proteins were separated on 10% SDS‐PAGE by electrophoresis and electro‐transferred to 0.45 μm PVDF membrane (Millipore). Antibodies used in this study were ERα (Invitrogen, MA5‐13304), HA (Proteintech, 51064‐2‐AP), Myc (Proteintech, 60003‐2‐Ig), USP37 (Proteintech, 18465‐1‐AP), GST (Proteintech, 66001‐2‐Ig), and GAPDH (Proteintech, 60004‐1‐Ig) antibodies.

Cao J., Wang X., Wang S., Chen Z, & Tang J. (2023). Stabilization of estrogen receptor α by USP37 contributes to the progression of breast cancer. Cancer Science, 114(5), 2041-2052.

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In Vivo Deubiquitination Assay for SLC7A11

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We performed in vivo deubiquitination assay as described in our previous study.
⁷⁶ (link) Briefly, HA‐Ub, Myc‐SLC7A11, Flag‐USP20, or Flag‐USP20^C154A plasmid were transfected into HEK293T cells for 48 h and treated with 10 μM MG132 (MCE) for another 6 h. Cells were harvested at 4°C using RIPA extraction reagent (Meilun) after washed three times with prechilled PBS. Ubiquitinated SLC7A11 was isolated and detected through Western blotting. In HCC cells, USP20 shRNAs and HA‐Ub plasmid were cotransfected into Huh‐7 cells. Ubiquitinated SLC7A11 was isolated and detected through Western blotting. Each experiment was repeated in triplicate at least three times.

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Immunoprecipitation and Immunoblotting Protocol

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Cells were washed with pre-chilled phosphate-buffered saline (PBS) and lysed with RIPA extraction reagent (Meilun, China) supplemented with protease inhibitors (Meilun, China). Cell lysates were pre-cleared and incubated with indicated antibody overnight at 4 °C, the antibody associated with the protein complex were then incubated with protein A/G PLUS-Agarose beads for additional 2 h. The beads were washed with PBS three times and boiled at 100 °C for 10 min to reverse crosslinking before SDS-PAGE immunoblotting analysis.

Wu L., Ou Z., Liu P., Zhao C., Tong S., Wang R., Li Y., Yuan J., Chen M., Fan B., Zu X., Wang Y, & Tang J. (2023). ATXN3 promotes prostate cancer progression by stabilizing YAP. Cell Communication and Signaling : CCS, 21, 152.

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Protein Co-Immunoprecipitation Assay

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Cells were washed with pre-chilled phosphate-buffered saline (PBS) and lysed with RIPA extraction reagent (Meilun, Dalian, China) supplemented with protease inhibitors (Meilun, Dalian, China). Cell lysates were pre-cleared and incubated with indicated antibody overnight at 4 °C, The antibody associated with the protein complex were then incubated with protein A/G PLUS-Agarose beads (beyotime, China) for additional 2 h. The beads were washed with PBS three times and boiled at 100 °C for 10 min to reverse crosslinking before SDS-PAGE immunoblotting analysis.

Tang J., Long G., Xiao L, & Zhou L. (2023). USP8 positively regulates hepatocellular carcinoma tumorigenesis and confers ferroptosis resistance through β-catenin stabilization. Cell Death & Disease, 14(6), 360.

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