TALE proteins were detected in Xoo strains by immunoblotting as described previously (Xu et al., 2019 (link)). In brief, Xoo strains were cultured in NB to the logarithmic phase and harvested by centrifugation. Bacterial cells were washed twice, and the concentration was adjusted to OD600 = 2.0 with sterile distilled water. SDS loading buffer (5×) was added to the bacterial suspensions; these were then boiled in a water bath for 10 min, separated on SDS–PAGE gels, and transferred to polyvinylidene difluoride membranes for immunoblotting with anti-FLAG (TransGen) as the primary antibody. Goat anti-rabbit immunoglobulin G (TransGen) was used for detection of primary antibodies with the EasySee Western Kit supplied by TransGen.
Easysee western kit
Manufactured by Transgene
Sourced in China
The EasySee Western Kit is a laboratory product that enables the detection and analysis of proteins using the western blotting technique. It provides the necessary reagents and components required to perform this analytical procedure.
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5 protocols using easysee western kit
1
TALE Protein Detection in Xoo Strains
2
TALE Protein Detection in Xoo Strains
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To detect the TALE proteins in Xoo strains, Western blotting was conducted as described previously [33] (link). The tested Xoo strains were cultured in NB to the logarithmic phase and harvested by centrifugation. Bacterial cells were washed twice, and adjusted to OD600 = 2.0 with sterile distilled water. Proteins added loading buffer were boiled, separated on an SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane for immunoblotting with anti-FLAG (TransGen, China) as the primary antibody. Then the goat anti-rabbit IgG (TransGen, China) was used to detect the primary antibodies. The blotting was visualized with the EasySee Western Kit supplied by TransGen (China).
3
Protein Expression Vector Construction and Characterization
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The protein expression vectors pH1-hrpG::FLAG, pH3-hrpX::FLAG, and pH3-hrpB1::FLAG were constructed in our previous study [6 (link)], then were electroporated into the Xoo PXO99A, PΔminC, PΔminD, and PΔminCDE, respectively. Overnight, Xoo strains were grown in NB medium at 28 °C and collected by centrifugation. Bacterial cells were rinsed with sterile water and resuspended at an OD600 of 2.0 in a type III-inducing XOM3 medium. These XOM3 suspensions were incubated in the shaken culture at 28 °C for 12 h. Protein samples were extracted from XOM3 suspension and separated by 10% SDS-PAGE. The proteins were then transferred to a PVDF membrane for immunoblotting using the Flag tag and anti-mouse IgG antibody (TransGen, Beijing, China). The membrane was visualized with the EasySee Western Kit (TransGen, Beijing, China). We used the E. coli RNA polymerase subunit (RNAP) antibody as the loading control.
4
Western Blot Analysis of Protein Expression
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At the end of this study, proteins were isolated from liver and eWAT using RIPA lysis buffer (Beyotime biotechnology, China) and protein concentrations were measured by BCA Kit (Thermo Fisher Scientific, USA). Equal amount of protein in each sample was separated by 10% SDS-PAGE and transferred to PVDF membranes (Biosharp, China). After blocked with 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase, respectively. After washing with PBS contained 0.05% Tween 20, the proteins were detected by EasySee Western Kit (Transgene, Beijing), and the blots were analyzed by ImageJ [46 (link)].
5
Secretion Detection of AvrXa7 and Tal7 Proteins
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Immunoblotting with Flag and c-Myc labeled antibodies was used to detect the secretion of AvrXa7-Flag and Tal7-c-Myc by Xoc YNB0-17. AvrXa7 was cloned in frame with C-terminal 3X FLAG-tag epitopes, and Tal7 and Tal7∆NA were cloned with C-terminal 3X c-Myc-tag epitopes. YNB0-17 bacterial cells containing the C-terminally tagged tal genes in constructs p707 and p707ΔNA (Supplemental Table S1 ) were cultured in NB to the logarithmic phase. The harvested bacteria were washed twice, and the OD600 was adjusted to 2.0 with sterile distilled water. The bacterial suspension (1 ml) was added to 40 ml filter-sterilized XOM3 medium61 and incubated at 28 °C for 6 h. The medium was centrifuged to separate total extracts (TE) and supernatant fractions (SN), and secreted proteins in the supernatant were precipitated with 12.5% trichloroacetic acid62 (link). Proteins were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes for immunoblotting using anti-FLAG or anti-C-Myc (Transgene, Beijing, China) as the primary antibody. Primary antibodies were detected using goat anti-rabbit IgG (H + L) (Transgene) and visualized with the EasySee Western Kit (Transgene).
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