For the pull-down assays, 3 μg bait and prey proteins were incubated overnight at 4°C. The beads were washed with a solution containing 20 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 separated on a sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and analyzed by immunoblotting using an anti-GST antibody (GenScript, Nanjing, China; A00866-100, lot: 18A001413) or an anti-His antibody (Abmart, Shanghai, China; M30111M, lot: 293871). Co-IP was performed as described previously (Su et al., 2017 (link)). Briefly, 1 × 106 protoplasts were lysed with PEN-140 buffer (140 mM NaCl, 2.7 mM KCl, 25 mM Na2HPO4, 1.5 mM KH2PO4, 0.01 mM EDTA and 0.05% CA-630). The supernatant was precipitated with anti-FLAG (Sigma-Aldrich; H8592, lot: SLBV3799) antibodies, followed by washes with PEN-400 buffer (400 mM NaCl, 2.7 mM KCl, 25 mM Na2HPO4, 1.5 mM KH2PO4, 0.01 mM EDTA and 0.05% CA-630). The samples were analyzed by immunoblotting using anti-HA (Roche; 11867423001, lot: 13500600) and anti-FLAG antibodies.
Anti his antibody
Manufactured by Abmart
Sourced in China, United States
The Anti-His antibody is a laboratory reagent used to detect and purify recombinant proteins containing a histidine tag. It specifically binds to the histidine tag, allowing for the identification and isolation of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Pan anti kcr antibody, by PTM Biolabs (2 mentions)
Magnet gst particles, by Promega (2 mentions)
Anti gst antibody, by Abmart (2 mentions)
Anti gst antibody, by GenScript (1 mentions)
Anti flag, by Roche (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Anti beta actin antibody, by Proteintech (1 mentions)
Anti calnexin antibody, by Proteintech (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Glutathione agarose beads, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 2, by GE Healthcare (1 mentions)
His tag purification resin, by Beyotime (1 mentions)
His tag protein purification kit, by Beyotime (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Glutathione agarose beads, by GE Healthcare (1 mentions)
Nickel affinity column, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Dna polymerase, by Thermo Fisher Scientific (1 mentions)
Gly pro p nitroanilide, by Merck Group (1 mentions)
22 protocols using anti his antibody
1
Protein-Protein Interaction Assays
2
Soybean Protein Degradation Assay
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Total proteins were extracted from WT and transgenic soybean lines with degradation buffer82 (link). Each reaction contained 500 µg of soybean total proteins and 100 ng of GmLHP1-His proteins purified from E. coli Rosetta (DE3) cells. For the proteasome inhibitor experiments, 100 µM MG132 was added to the total proteins 60 min prior to the cell-free degradation experiment. The reactions were incubated at 22 °C. The mixed solutions were collected at the designated time point (0, 0.5, 1, and 3 h) and examined using an anti-His antibody (Abmart, code number M20001S). The quantified results were analyzed using ImageJ software (https://imagej.nih.gov/ij/index.html ).
3
Protein Expression and Detection
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Cell culture medium was obtained from life technologies (Staley Rd Island, USA), and FBS was obtained from Pan seratech (Aidenbach, Bavaria, Germany). EDTA-free protein inhibitor cocktail was purchased from Bimake (Houston, USA), Polyethylenimine (PEI) were obtained from Polysciences (Warrington, PA).
The following primary antibodies were used: Anti-ANGPTL3 antibody (AF136, R&D Systems, Minneapolis, USA), Anti-His antibody (Catalog: M20008F, Abmart, Berkeley Heights, US), Anti-Beta actin antibody (Catalog: 20536-1-AP, Proteintech, Chicago, IL), Anti-Calnexin antibody (Catalog: 10427-2-AP, Proteintech, Chicago, IL). The following secondary antibodies were used: HRP conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch), bovine anti-goat IgG (Proteintech, Chicago, IL).
The following primary antibodies were used: Anti-ANGPTL3 antibody (AF136, R&D Systems, Minneapolis, USA), Anti-His antibody (Catalog: M20008F, Abmart, Berkeley Heights, US), Anti-Beta actin antibody (Catalog: 20536-1-AP, Proteintech, Chicago, IL), Anti-Calnexin antibody (Catalog: 10427-2-AP, Proteintech, Chicago, IL). The following secondary antibodies were used: HRP conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Jackson ImmunoResearch), bovine anti-goat IgG (Proteintech, Chicago, IL).
4
Evaluating TaSRT1 Decrotonylation of TaPGK
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To test whether TaSRT1 can decrotonylate TaPGK in vitro, TaPGK-His and TaSRT1-GST or GST vector were transformed in E. coli. We purified TaPGK-His recombinant protein from E. coli. The purified proteins were detected by immunoblot using anti-His antibody (Abmart, M30111) and pan anti-Kcr antibody (PTM Biolabs, lot number 502).
5
Protein-Protein Interaction Assays Using Fusion Proteins
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To construct GST-fusion plasmids, MoARK1 were inserted into the vector pGEX4T-2 (GE Healthcare Life Science). To construct His-fusion plasmid, MoCAPA and MoCAPB were inserted into the vector pET-32a (Navogen), respectively. Pull-down assay was carried out using profound pull-down GST protein-protein interaction kit (Pierce) according to the manufacturer’s instructions. Briefly, GST or GST-MoARK1 was expressed in E. coli strain BL21 (DE3). Soluble proteins were incubated with 50 μl glutathione agarose beads (Invitrogen) for 2 h at 4˚C. The beads were washed five times and then incubated with an equal amount of bacterial lysates containing His-MoCapA or His-MoCapB for another hour at 4˚C. The beads were washed five times again, and the presence of His-MoCapA or His-MoCapB was detected by immuno-blotting using anti-His antibody (Abmart).
6
Expression and Purification of Recombinant TaPGK-His
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TaPGK was cloned into the prokaryotic expression vector PET28a using Bam HI and Not I as restriction enzyme cutting sites (table S1). E. coli BL21 (DE3) was used to express recombinant TaPGK-His. The elution buffer [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 (pH 7.4), and 0.2 mM DTT] was used to elute the proteins using BeyoGold His-tag Purification Resin according to the instruction of the His-tag Protein Purification Kit (Beyotime, China, catalog no. P2226), then the collected TaPGK-His recombinant proteins were analyzed by SDS-PAGE and immunoblot by using anti-His antibody (Abmart, M30111) and pan anti-Kcr antibody (PTM Biolabs, lot number 502), respectively.
7
Affinity Purification of NUO-RIR1 Complex
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Recombinant His‐NUO, MBP or MBP‐RIR1‐K were expressed in E. coli BL21 (DE3) for use in the pull‐down assays. Co‐precipitation of NUO containing the RIR1 kinase domain was examined by western blotting after affinity purification using amylose resin (NEB) following the manufacturer's instructions. Total proteins were extracted in binding buffer (20 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1× protease inhibitor cocktail [Roche]) at 4 °C, and then coprecipitated with NUO with RIR1‐K in the binding buffer. After incubation, the resin was washed five times with washing buffer (20 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1× protease inhibitor cocktail [Roche]) and eluted in 20 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM amylose. The presence of His‐NUO in the eluate was detected with an anti‐HIS antibody (Abmart).
8
In Vitro Protein-Protein Interaction Assay
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N-terminal His-tagged GmSNAP-1 and MBP-tagged PsAvh181 were expressed in E. coli (strain BL21). Purified proteins were dissolved in 1× phosphate-buffered saline (PBS) buffer with 1 mM phenylmethylsulfonyl (PMSF). His-GmSNAP was incubated with Ni-NTA agarose for 2 h at 4°C. Beads were added to the total protein extracted from the supernatant of E. coli expressing MBP-PsAvh181 and incubated for 3 h at 4°C. The Ni-NTA agarose was washed four times using 1× PBS buffer. Proteins were eluted by boiling SDS loading buffer for 5 min, and then analyzed by SDS-PAGE and western blot analysis. His-GmSNAP was detected using an anti-His antibody (Abmart, Shanghai, China). MBP-PsAvh181 was detected using an anti-MBP antibody (CMCTAG).
9
GST Pull-Down Assay for RXLR Effector Interaction
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We expressed GST, GST‐RXLR242, and NbRABE1‐7‐HIS in Escherichia coli Rosetta D36E and purified the proteins. For GST pull‐down, 2 ml of the total protein extract containing GST or GST‐PcRXLR242 was incubated with 30 μl of glutathione agarose beads (GE Healthcare) at 4°C for 4 h with rotation. The mixture was thoroughly washed twice or three times with TBS buffer containing 0.1% Triton‐X‐100. After removing the supernatant, the beads were added to the total protein extract containing NbRABE1‐7‐HIS and incubated for an additional 4 h at 4°C. The pull‐down of RABE1‐7‐HIS was detected using an anti‐His antibody (Abmart).
10
DPP Enzymatic Activity Assay
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The recognised dipeptidyl peptidase (DPP) substrate Gly-Pro-p-nitroanilide and DPP4 enzyme were purchased from Sigma (Missouri, USA). The VigoScript first strand cDNA synthesis kit was a product of Vigorous (Beijing, China). Rosetta competent cells were obtained from TransGen (Beijing, China). Restriction enzymes were purchased from Takara (Dalian, China). Nickel affinity columns, Trizol reagent and DNA polymerase were obtained from Invitrogen (Shanghai, China). The plasmid vector pET32-a(+) was a product of Novagen (Shanghai, China). The anti-His antibody was purchased from Abmart (Shanghai, China). The DPP inhibitor compounds, sitagliptin and UAMC00132 were provided by Prof. Haihong Huang.
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