Ht601

Manufactured by Transgene

The HT601 is a laboratory equipment product designed for high-throughput sample processing. It is a versatile instrument that can handle a variety of sample types and volumes. The core function of the HT601 is to automate and streamline sample preparation workflows, improving efficiency and consistency in laboratory operations.

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Lab products found in correlation

Ht501, by Transgene (3 mentions) Coomassie brilliant blue r 250, by Avantor (2 mentions) Hrp conjugated goat anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions) Glutathione sepharose 4b, by GE Healthcare (1 mentions) Ht101, by Transgene (1 mentions) Clonexpress 2 one step cloning kit, by Vazyme (1 mentions) Glutathione sepharose 4b matrix, by GE Healthcare (1 mentions)

7 protocols using ht601

In vitro Pull-down Assay for ABA Signaling Proteins

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The in vitro pull-down assay was performed as previously described^{76 (link)}. Briefly, 5 μg of purified GST-PYR1/PYLs recombination protein was incubated with GST-Sefinose (TM) Resin 4FF (Settled Resin) (BBI) at 4 °C for 2 h and incubated with 2 μg of CIPK1-His for another 2 h in a pull-down buffer, after which the beads were washed 6 times using the pull-down buffer. The proteins were separated by SDS-PAGE for the immunoblot assay using anti-His (1:5000, TransGen, HT501) antibody and anti-GST (1:5000, TransGen, HT601) antibody. The interactions between ABI1 and PYLs were also determined in pull-down assays.
For ABA inhibition of CIPK1 interaction with PYLs, 5 μg of CIPK1-His protein was incubated on Ni-NTA beads for 2 h and incubated with 2 μg of GST-PYL1 or GST-PYL4 for another 2 h in the pull-down buffer. Then the beads were washed 6 times using the pull-down buffer. The proteins were separated by SDS-PAGE for the immunoblot assay using anti-His (1:5000, TransGen, HT501) antibody and anti-GST (1:5000, TransGen, HT601) antibody.

You Z., Guo S., Li Q., Fang Y., Huang P., Ju C, & Wang C. (2023). The CBL1/9-CIPK1 calcium sensor negatively regulates drought stress by phosphorylating the PYLs ABA receptor. Nature Communications, 14, 5886.

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GST-HlgB Protein-Protein Interaction Assay

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The 96-well plates were coated with YG8–1 overnight. After blocking with 5% non-fat milk at 37 °C for 1 h, serially dilutions of YG8–2 and YG8–3 were added to the wells respectively, which mixed with 10 μg/mL GST-HlgB, after incubated at 37 °C for 1 h and washed 5 times with PBST, the monoclonal mouse anti-GST antibodies (Transgene, HT601, 1:1000) were added for 45 min at 37 °C. Followed by the addition of a HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, 115–035-174, 1:20000) for 30 min at 37 °C. The plates were washed 3 times and incubated for 30 min with TMB. Optical density at 450 nm was measured in a microtiter plate reader.

Jing C., Liu C., Liu F., Gao Y., Liu Y., Guan Z., Xuan B., Yu Y, & Yang G. (2018). Novel human monoclonal antibodies targeting the F subunit of leukocidins reduce disease progression and mortality caused by Staphylococcus aureus. BMC Microbiology, 18, 181.

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Affinity Purification of Fusion Proteins

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The fusion proteins were purified as described previously (Mao et al., 2014). Then, equivalent protein of His‐MoBre2_FL and GST‐MoSdc1_FL fusion proteins were added to a 1.5 ml centrifuge tube with BC100 (20 mM Tris.HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.1% NP‐40) to 400 μl. Next, 40 μl glutathione‐sepharose 4B (GE Healthcare) was added to the solution. The tubes were then rotated and incubated for 4 hr at 4 °C to pull down GST‐tagged fusion proteins. This was followed by four washes with BC500 (20 mM Tris.HCl [pH 8.0], 500 mM NaCl, 1 mM EDTA, 0.1% NP‐40). The beads were boiled in SDS loading buffer for 10 min, centrifuged at 1,575 × g at 4 °C for 5 min, and then the supernatant was separated by 13% denaturing SDS‐PAGE. Finally, the gel was stained with Coomassie Brilliant Blue R‐250 (AMRESCO) and western blotting using anti‐His (Transgen Biotech, HT501) and anti‐GST antibodies (Transgen Biotech, HT601) (Mao et al., 2017) for observation.

Zhou S., Liu X., Sun W., Zhang M., Yin Y., Pan S., He D., Shen M., Yang J., Zheng Q, & Wang W. (2021). The COMPASS‐like complex modulates fungal development and pathogenesis by regulating H3K4me3‐mediated targeted gene expression in Magnaporthe oryzae. Molecular Plant Pathology, 22(4), 422-439.

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Investigating OsRpp30 Acetylation in E. coli and Rice

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For the experiments in E. coli, GST‐OsRpp30 and His‐OsRpp30 expressed in E. coli, grown without or with 10 nm TSA, were first isolated by immunoprecipitation with glutathione (Abcam, #ab193267) and nickel (Qiagen, #30210) sepharose beads, respectively. In subsequent immunoblotting, the antibody against GST (TransGen, #HT601) or the poly‐His tag (TransGen, #HT501) was used to assess the amount of pulled down OsRpp30 fusion proteins, and a pan acetylation monoclonal antibody (Proteintech, Rosemont, IL, #66289‐1‐Ig) was used to detect OsRpp30 acetylation. For the experiment in rice protoplasts, the OsRpp30‐Myc plasmid alone or together with the HDT701‐Flag plasmid was transfected into protoplasts of the indicated genotypes. Subsequently, OsRpp30‐Myc was purified using anti‐Myc beads for use in immunoblotting. A Myc antibody (TransGen, #HT101) was used to assess OsRpp30‐Myc amount in each lane, and the acetylation antibody was used to determine OsRpp30‐Myc acetylation.

Li W., Xiong Y., Lai L.B., Zhang K., Li Z., Kang H., Dai L., Gopalan V., Wang G, & Liu W. (2021). The rice RNase P protein subunit Rpp30 confers broad‐spectrum resistance to fungal and bacterial pathogens. Plant Biotechnology Journal, 19(10), 1988-1999.

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Protein-Protein Interaction Assay

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The OsDLA coding sequence was cloned into the pGEX‐4T‐1 vector, and the coding sequences of OsWRKY53 were cloned into pMAL‐C4X. ClonExpress II One Step Cloning Kit (C112, Vazyme Biotech) was used for subcloning, and the recombinant proteins were expressed and purified from E. coli strain BL21 (DE3). Pull‐down assays were performed according to the method described by Meng et al. (2020 (link)). MBP‐ and GST‐fused recombinant proteins were detected by immunoblotting using an anti‐MBP antibody (TransGen Biotech, HT701) and an anti‐GST antibody (TransGen Biotech, HT601), respectively.

Meng F., Zheng X., Wang J., Qiu T., Yang Q., Fang K., Bhadauria V., Peng Y, & Zhao W. (2023). The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice. Plant Biotechnology Journal, 22(2), 363-378.

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Purification of GST-Kindlin-2 and PYCR1

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For generation of GST-fusion proteins containing wild type or mutant forms of kindlin-2, the corresponding cDNA sequences were cloned into pGEX-4T-1 vector. GST and GST-fusion proteins were expressed in Escherichia coli strain BL21 (DE3) cells and purified with Glutathione-Sepharose 4B matrix (GE Healthcare) following the manufacturer’s protocol. PYCR1 was expressed with His tag at the N-terminal in E. coli and purified with nickel resin (Sangon). Purified proteins were resolved by SDS-PAGE to verify their size and purity. In pulldown assays, GST or GST-fusion proteins bound to glutathione-Sepharose beads were incubated with purified His-tagged PYCR1 for 2 h at 4 °C. The beads were washed three times with buffer (20 mM Tris pH 7.5, 150 mM NaCl and 0.1% Triton) and analyzed by SDS-PAGE. His- and GST-tagged proteins were detected by western blotting with mouse anti-His (Tiangen, AB102, 1:1000) and anti-GST (Transgen, HT601, 1:1000) antibodies, respectively.

Guo L., Cui C., Zhang K., Wang J., Wang Y., Lu Y., Chen K., Yuan J., Xiao G., Tang B., Sun Y, & Wu C. (2019). Kindlin-2 links mechano-environment to proline synthesis and tumor growth. Nature Communications, 10, 845.

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Purification and Analysis of Fusion Proteins

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The fusion proteins were purified as described previously (Mao et al., 2014) . Then, equivalent protein of His-MoBre2 FL and GST-MoSdc1 FL fusion proteins were added to a 1.5 mL centrifuge tube and the solution was brought to 400 μL by BC100 (20 mM Tris-HCl [pH=8.0], 100 mM NaCl, 1 mM EDTA, 0.1% NP-40). Next, 40 μL
Glutathione-Sepharose 4B (GE Healthcare, 17-0756-01) was added to the solution.
The tubes were then rotated and incubated for 4 h at 4 °C to pull down GST-tagged fusion proteins. This was followed by four washes with BC500 (20 mM Tris-HCl [pH=8.0], 500 mM NaCl, 1 mM EDTA, 0.1% NP-40). The beads were boiled in SDS loading buffer for 10 min, centrifuged at 3500 rpm at 4 °C for 5 min, and then the supernatant was separated by 13% denaturing polyacrylamide gel (SDS-PAGE).
Finally, the gel was stained with Coomassie Brilliant Blue R-250 (AMRESCO, 0472-10G) and Western blotting using anti-His (Transgen Biotech, HT501) and anti-GST antibodies (Transgen Biotech, HT601) for observation.

Zhou S., Sun W., Zhao X., Xu Y., Zhang M., Yin Y., Pan S., He D., Shen M., Yang J., Liu X., Zheng Q., & Shang Y. (2020). The COMPASS-like complex modulates fungal development and pathogenesis by regulating H3K4me3-mediated targeted gene expression in Magnaporthe oryzae.

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