The antibodies used in this study were as follows: anti-C1QTNF5, anti-Lamin B1, anti-Na+/K+-ATPase, anti-Myc, anti-β-Actin, anti-GAPDH, anti-GST and anti-His (A3021, A1910, A11683, AE070, AC026, AC002, AE001 and AE003, respectively; ABclonal, China); anti-α-Tubulin, anti-Flag and anti-HA (T6199, F1804 and H6908, respectively; Sigma); anti-NP (ab128193; Abcam); anti-H1N1 HA (11684-MM03; Sino Biological); goat anti-mouse or anti-rabbit IgG (115-035-174 and 111-005-144; Jackson Immuno Research Laboratories); goat anti-mouse antibodies conjugated with Alexa Fluor 488 (A-11001; Invitrogen). The main reagents used in this study were as follows: anti-Flag M2 beads, TPCK, sialidase and blasticidin (A2220, T1426, N7885 and 203350, respectively; Sigma); Lipofectamine 2000 (11668019; Thermo Fisher Scientific); Dual luciferase reporter assay kit (E1980; Promega); Neofect™ DNA transfection reagent (TF20121201; Neofect); Minute™ plasma membrane protein and cell component separation kit (SM-005; Invent Biotechnologies).
Ae001
Manufactured by ABclonal
Sourced in China
AE001 is a laboratory equipment designed for protein extraction. It provides a controlled environment for the efficient isolation and purification of proteins from a variety of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ae003, by ABclonal (3 mentions)
A2220, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
N7885, by Merck Group (1 mentions)
H6908, by Merck Group (1 mentions)
T1426, by Merck Group (1 mentions)
Ac026, by ABclonal (1 mentions)
Anti lamin b1, by ABclonal (1 mentions)
Anti na k atpase, by ABclonal (1 mentions)
Anti myc, by ABclonal (1 mentions)
A1910, by ABclonal (1 mentions)
F1804, by Merck Group (1 mentions)
T6199, by Merck Group (1 mentions)
Anti gst, by ABclonal (1 mentions)
Anti flag m2 bead, by Merck Group (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Ac002, by ABclonal (1 mentions)
Ab55268, by Abcam (1 mentions)
6 protocols using ae001
1
Antibody-based Target Identification Workflow
2
Antibody Panel for 3βHSD1 and BMX Phosphorylation
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Mouse monoclonal antibodies against 3βHSD1 (1:2,000, ab55268) and rabbit polyclonal antibodies against phospho-BMX (1:2,000, ab59409) were purchased from Abcam. Mouse monoclonal antibodies against phospho-tyrosine (pTyr) (1:2,000, 05-1050) were purchased from Sigma-Aldrich. custom-made rabbit monoclonal antibodies against phospho-3βHSD1 Y344 (1:2,000) were ordered from Affinity Biosciences. Mouse monoclonal antibodies against GST (1:5,000, AE001) were purchased from Abclonal. Mouse monoclonal antibodies against Flag (1:5,000, F3165) and anti-Flag M2 affinity gel (A2220) were purchased from Sigma-Aldrich. Rabbit monoclonal antibodies against HA (1:3,000, 3724S), β-actin (1:3,000, 3700S), and rabbit polyclonal antibodies against BMX (1:3,000, 24773) and GAPDH (1:5,000, 14C10) were obtained from Cell Signaling Technology.
3
Protein Pull-Down Assay Protocol
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All proteins used in this study were expressed in Escherichia coli BL21 (induced with 0.5 mm IPTG at 16 °C overnight) and purified using glutathione beads (Smart-Lifesciences, SA008100) or Ni-IDA Beads (Smart-Lifesciences, SA003025). The GST or GST-WEE1 proteins coupled to glutathione beads were incubated with GCN20-HIS in binding buffer (140 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 , and 1.8 mm KH 2 PO 4 , pH 7.4) for 2 h at 4 °C. The beads were washed 3 times with washing buffer (140 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 , 1.8 mm KH 2 PO 4 , and 1% Triton X-100, pH 7.4). The proteins were eluted by incubating the beads in 2 × SDS loading buffer at 100 °C for 8 min. Both the input and pull-down samples were subjected to immunoblotting using anti-GST (1:4,000, Abclonal, AE001) or anti-HIS (1:4,000, Abclonal, AE003) antibodies.
4
Phosphorylation Assay of HIS-GCN20 Proteins
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The HIS-GCN20 proteins were incubated with GST-WEE1 or GST-WEE1 kd in phosphorylation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm MgCl 2 , 0.1% Nonidet P-40, 0.1% Triton X-100, 500 µm ATP, and 1 mm PMSF) at 30 °C for 1 h. The reactions were stopped by adding 5 × SDS loading buffer and subjected to SDS-PAGE using gels with or without 25 μm Phos-Tag (Wako, 300-93523). The proteins were subjected to immunoblotting using anti-GST (1:3,000, Abclonal, AE001) and anti-HIS (1:3,000, Abclonal, AE003) antibodies.
5
Immunoblotting of Recombinant Proteins
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Arabidopsis seedlings were homogenized in proteolysis buffer (25 mm Tris-HCl, pH 7.5, 10 mm MgCl 2 , 10 mm NaCl, 10 mm ATP, and 5 mm DTT) at 4 °C and centrifuged at 12,000 × g for 15 min. The recombinant proteins were incubated with the supernatants at room temperature for different amounts of time. The reactions were stopped by adding 2 × SDS loading buffer and boiled at 100 °C for 8 min. The samples were subjected to immunoblotting using anti-GST (1:3,000, Abclonal, AE001), anti-HIS (1:3,000, Abclonal, AE003), or anti-GS (1:4,000, Agrisera, AS08295) antibodies.
6
Protein-Protein Interaction Analysis via Pull-Down Assay
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For in vitro pull-down assays, His-tagged BnWRKY33, BnaA03.MKK4, and BnaA09.VQ12, and GST-tagged BnaA06.MPK3, BnaC03.MPK3, BnaA03.WRKY28, and BnaA03.WRKY28 164-227 proteins were used. GST or GST-tagged protein sample was incubated with GST beads at 4°C for 1h, followed by washing five times, and incubation with His-tagged protein at 4°C
for 2 h. The beads were then washed five times. The isolated precipitate was boiled with loading buffer at 100°C for 6 min and detected by western blot using anti-His (Abclonal, AE003, China, 1:10000) or anti-GST (Abclonal, AE001, China, 1:10000) antibodies. The ECL western specific luminescence detection kit (BIO-RAD, #1705060, USA) was used to collect the signal, and the images were scanned using the Image Quant luminescence LAS 4000 machine.
for 2 h. The beads were then washed five times. The isolated precipitate was boiled with loading buffer at 100°C for 6 min and detected by western blot using anti-His (Abclonal, AE003, China, 1:10000) or anti-GST (Abclonal, AE001, China, 1:10000) antibodies. The ECL western specific luminescence detection kit (BIO-RAD, #1705060, USA) was used to collect the signal, and the images were scanned using the Image Quant luminescence LAS 4000 machine.
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