Gerpn1210b

Manufactured by Merck Group

The GERPN1210B is a laboratory equipment product manufactured by the Merck Group. It is a precision instrument designed for specific laboratory applications. The core function of this product is to perform accurate measurements and data collection in a controlled laboratory environment. No further details or interpretations about the intended use of this product can be provided.

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Lab products found in correlation

Trizol, by Thermo Fisher Scientific (2 mentions) 3 3 diaminobenzidine peroxidase substrate kit, by Yeasen (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) M6a methylation antibody, by Synaptic Systems (1 mentions) Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions) Ab252215, by Abcam (1 mentions) Chemiluminescent reagent, by Merck Group (1 mentions) Horseradish peroxidase conjugated anti rabbit immunoglobulin g, by Cell Signaling Technology (1 mentions) Rabbit anti m6a antibody, by Synaptic Systems (1 mentions) Anti m6a antibody, by Synaptic Systems (1 mentions) Immobilon western hrp substrate, by Merck Group (1 mentions)

6 protocols using gerpn1210b

Detecting m6A RNA Methylation Levels

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Total RNA was extracted, and 100 ng of RNA was spotted onto a nylon membrane (Sigma-Aldrich, GERPN1210B). Then, the membranes were UV-crosslinked and blocked with PBST (PBS with 0.1% Tween 20) containing 5% nonfat milk for 1 h. After that, the membranes were incubated with m6A methylation antibody (Synaptic Systems, 202003, 1:1000) at 4 °C overnight. Then, the membranes were washed with PBST and incubated with HRP-conjugated secondary antibodies (1:3,000, Boster, BA1054) for 1 h at room temperature. The membranes were washed with PBST and detected by using Immobilon Western Chemiluminescent HRP Substrate (Millipore WBKLS0500).
The same amount of RNA was spotted on another nylon membrane, and UV crosslinking was performed. Then, the membranes were stained with 0.02% methylene blue solution (pH 5.2) for 1 h. The membranes were washed with ribonuclease-free water for 2 h, and the results were captured by a camera.

Ye G., Li J., Yu W., Xie Z., Zheng G., Liu W., Wang S., Cao Q., Lin J., Su Z., Li D., Che Y., Fan S., Wang P., Wu Y, & Shen H. (2023). ALKBH5 facilitates CYP1B1 mRNA degradation via m6A demethylation to alleviate MSC senescence and osteoarthritis progression. Experimental & Molecular Medicine, 55(8), 1743-1756.

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Quantifying m6A RNA Modifications

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The m6A content in the poly-A tailing of total RNA was measured using an RNA ac4C dot blot assay. Total RNA was extracted using TRIzol (Invitrogen, 15,596,018) according to the manufacturer's guidelines. After double dilution, the RNAs (300 ng) were applied onto a nylon membrane (Sigma-Aldrich, GERPN1210B). The membranes underwent ultraviolet crosslinking and were then blocked using 5 % nonfat milk. Afterward, they were incubated overnight with the anti-ac4C antibody (Abcam, ab252215). The membranes were then incubated with the secondary antibody for 1 h at room temperature. The Tanon 5800 image system (Tanon, Shanghai, China) was used to detect signals. To demonstrate the quantity of total RNA, a solution of 0.02 % methylene blue in 0.3 M sodium acetate (pH 5.2) was employed.

Shen J., Sun Y., Zhuang Q., Xue D, & He X. (2024). NAT10 promotes renal ischemia-reperfusion injury via activating NCOA4-mediated ferroptosis. Heliyon, 10(2), e24573.

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RNA Dot Blot Analysis of m6A

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Total RNA was extracted from the cells using TRIzol and the RNA concentration was determined by a NanoDrop 2000 (Thermo Fisher Scientific). The RNA concentration of different samples was adjusted to 250 and 500 ng/µl. Two microliters of RNA was spotted onto the nylon membrane (Sigma-Aldrich, GERPN1210B), followed by 1500 J ultraviolet crosslinking twice. One of the membranes was dyed in methylene blue solution for 30 min. After 30 min, the membrane was rinsed twice with ultrapure water and photographed. The other membrane was blocked with 5% nonfat dry milk dissolved in PBST (PBS with 0.1% Tween-20) for 1 h, followed by incubation with m⁶A antibody (1 : 1000, 202003, Sysy) for 14–16 h. Then, PBST was added to wash the membrane for 5 min, which was repeated three times. An horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG dilution (1 : 3000, BA1050, BOSTER) was added and the membrane was incubated at room temperature for 1 h. Then, PBST solution was added again to rinse the membrane three times. The protein signals were detected using chemiluminescent reagents (Millipore) according to the manufacturer’s instructions.

Li Z., Wang P., Li J., Xie Z., Cen S., Li M., Liu W., Ye G., Zheng G., Ma M., Wang S., Yu W., Wu Y, & Shen H. (2021). The N6-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6. Cell Death & Disease, 12(6), 578.

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Dot Blot Analysis of m6A RNA Modifications

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Dot blots were performed as follows. Total RNA was isolated. The RNAs (100 and 250 ng respectively) were double diluted and spotted onto a nylon membrane (Sigma-Aldrich, GERPN1210B). Then the membranes were ultraviolet (UV) crosslinked and blocked in blocking buffer (5% milk in phosphate-buffered saline with 0.1% Tween 20) for 1 h. Rabbit anti-m⁶A antibody (Synaptic Systems, 202003) was diluted 1:1,000 and incubated with the membranes overnight (4°C). After washing twice with 0.1% phosphate-buffered saline-Tween 20, horseradish peroxidase conjugated anti-rabbit immunoglobulin G (Cell Signaling Technology, 7074S) was diluted 1:5,000 and incubated with the membranes for 1 h at room temperature. After extensive washing, membranes were detected with a 3,3′-diaminobenzidine peroxidase substrate kit (Yeasen Biotechnology, 36302ES01). The same amount of poly(A)⁺ RNAs were spotted on the membrane, stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) for 2 h, and washed with ribonuclease-free water for 5 h.

Song H., Feng X., Zhang H., Luo Y., Huang J., Lin M., Jin J., Ding X., Wu S., Huang H., Yu T., Zhang M., Hong H., Yao S., Zhao Y, & Zhang Z. (2019). METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes. Autophagy, 15(8), 1419-1437.

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m6A RNA Immunoprecipitation on Membrane

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Total RNA was purified using TRIzol (Cat No. 15596018, Invitrogen). RNAs (100 and 250 ng) were placed on a nylon membrane (Cat No. GERPN1210B, Sigma-Aldrich) prior to ultraviolet crosslinking and blocking (5% BSA) for 60 min. An anti-m⁶A antibody (1:1000, Cat No. 202003, Synaptic Systems) was cultivated overnight (4 °C) with the membranes. Following rinsing with 0.1% phosphate-buffered saline-Tween 20, samples were exposed to secondary antibody for 60 min at room temperature. Following rinsing, membranes were probed using a 3,3′-diaminobenzidine peroxidase substrate kit (Cat No. 36302ES01, Yeasen Biotechnology). Equal quantity of RNAs was added onto the membrane, before methylene blue staining to display total RNA level [35 (link)].

Wu L., Du Y., Wang L., Zhang Y, & Ren J. (2024). Inhibition of METTL3 ameliorates doxorubicin-induced cardiotoxicity through suppression of TFRC-mediated ferroptosis. Redox Biology, 72, 103157.

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Quantifying m6A RNA Methylation

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The dot blot assay was performed as previously described [38 (link)]. Briefly, 100 ng of RNA from each sample was added to two nylon membranes (Sigma–Aldrich, GERPN1210B), and UV crosslinking was performed. One of the membranes was stained with methylene blue solution and imaged with a camera. The other membrane was blocked with 5% nonfat milk for 1 h at room temperature. Subsequently, the membrane was incubated with m6A methylation antibody overnight at 4 °C. Then, the membrane was washed and incubated with secondary antibodies for 1 h at room temperature. Finally, the membrane was washed and visualized by Immobilon Western HRP Substrate (Millipore, WBKLS0500).

Huang X., Huang X., Guo H., Li J., Zhou C., Huang Y., Lai C., Zeng W., Tan X., Niu L., Li H., Qi J, & Xie C. (2022). Intermittent hypoxia-induced METTL3 downregulation facilitates MGLL-mediated lipolysis of adipocytes in OSAS. Cell Death Discovery, 8, 352.

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