N. benthamiana leaf chloroplast and chloroplast-excluded fraction were separated as described (35 ). Leaves weighing 0.1 g were homogenized in 400 μL ice-cold isolation buffer (0.33 M Sorbitol, 50 mM Hepes pH = 7.0, 0.1% bovine serum albumin [BSA], 2 mM EDTA, 1 mM MgCl2) using a precooled glass homogenizer. The homogenate was filtered through a 50-μm mesh filter and kept on ice and in the dark for 5 min. Filtered chloroplast-containing homogenate (400 μL) was placed carefully on top of the 40% Percoll layer and centrifuged at 1,700 g at 4 °C for 6 min. The intact chloroplasts will sediment as a green pellet, whereas the chloroplast excluded fraction remains on the top. Chloroplasts were shock-frozen in liquid nitrogen and resuspended in 50 μL of ice-cold protein extraction buffer and centrifuged at 15,000 g at 4 °C for 10 min. The supernatant was extracted for Western blot. Anti-RbcL (Beijing Protein Innovation, AbP80037-A-SE) and anti-Act11 (Abmart, M20009) are used to detect chloroplast fraction and chloroplast-excluded fraction, respectively.
M20009
Manufactured by Abmart
Sourced in China
The M20009 is a laboratory equipment designed for general laboratory use. It serves as a basic tool for various scientific and research applications. The core function of this product is to provide a reliable and versatile platform for conducting laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
M20001, by Abmart (1 mentions)
Ripa strong lysis buffer, by Beyotime (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Supersignal west femto trial kit, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
As10 681, by Agrisera (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Ab97020, by Abcam (1 mentions)
M20008, by Abmart (1 mentions)
Supersignal western detection reagents, by Thermo Fisher Scientific (1 mentions)
Bcip nbt kit, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Abmart (1 mentions)
Phosstop, by Roche (1 mentions)
M20004, by Abmart (1 mentions)
Plant total protein extraction kit, by Merck Group (1 mentions)
As152894, by Agrisera (1 mentions)
As08 348, by Agrisera (1 mentions)
Anti osfd1 antibody, by ABclonal (1 mentions)
Psr 45, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
8 protocols using m20009
1
Chloroplast Isolation and Fractionation
2
In vitro Protein Degradation Assay for Arabidopsis
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The in vitro protein degradation assay was performed as described [58 (link)]. Leaves from 4-week-old Arabidopsis were ground in liquid N2 and resuspended in the proteolysis buffer (20 mM Tris [pH 7.5], 10 mM MgCl2, 10 mM NaCl, 10 mM ATP, and 5 mM DTT). After centrifugation, the supernatants from Arabidopsis were mixed with EBP1-His protein (S2C Fig ) and incubated at room temperature for 30 minutes. The reactions were stopped by boiling in SDS-PAGE sample buffer. Immunoblot assay was performed to detect the protein. Anti-His (M20001, Abmart) or anti-Actin (M20009, Abmart) were used to detect EBP1-His or Actin, respectively.
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from the roots of the sample using the RIPA lysis buffer (strong) (Beyotime, China). For Western blot analysis, proteins were electroblotted from 10% acrylamide gel to nitrocellulose membrane (Immobilon‐P, MILLIPORE Corporation, Bedford, MA, USA) after the separation of SDS‐PAGE. Antibodies used in Western blot were as follows: anti‐GFP antibody (M20004, Mouse mAb, Abmart, Shanghai, China), 1 : 1000 for Western blot; anti‐ACTIN antibody (M20009, Mouse mAb, Abmart, Shanghai, China), 1 : 1000 for Western blot and goat anti‐mouse lgG‐HRP (M21001, Abmart, Shanghai, China), 1 : 5000 for Western blot. Image Quant LAS 4000 (GE, USA), as the CCD camera system, was used for the band intensity quantification with Super Signal West Femto Trial Kit (Thermo, Rockford, IL, USA).
4
Immunoblotting of Stress-Induced Proteins
5
Western Blot Analysis of Fusion Proteins
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To examine the protein levels of FLAG- and GFP-tagged proteins, ∼100 mg of plant tissues were frozen in liquid nitrogen, ground thoroughly, and homogenized in 100-μL protein extraction buffer [20-mM Tris–HCl, pH 7.5, 150-mM NaCl, 0.5% (v/v) Tween-20, 1-mM EDTA, 1-mM 1,4-dithiothreitol (DTT)] containing a protease inhibitor cocktail (cOmplete, Roche) and a protein phosphatase inhibitor tablet (PhosSTOP, Roche). After addition of sodium dodecyl sulfate loading buffer, the samples were heated at 65°C for 5 min, resolved by 10% (v/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride membranes. FLAG-tagged proteins were detected by a mouse anti-FLAG antibody (M20008, 1:2,000, Abmart). GFP-tagged proteins were detected with a mouse anti-GFP antibody (M20004, 1:2,000, Abmart) or a mouse anti-GFP HRP (horseradish peroxidase)-conjugated antibody (130-091-833, 1:2,000, MACS Molecular). Actin was detected by a mouse anti-actin antibody (M20009, 1:2,000, Abmart).
After incubation with a primary mouse antibody, the PVDF membrane was then incubated with a goat anti-mouse immunoglobulin G AP-conjugated secondary antibody (ab97020, 1:5,000, Abcam). AP activity was detected by BCIP/NBT kit (Invitrogen) according to the supplier’s instructions. HRP activity was detected by the Supersignal Western Detection Reagents (Thermo Scientific).
After incubation with a primary mouse antibody, the PVDF membrane was then incubated with a goat anti-mouse immunoglobulin G AP-conjugated secondary antibody (ab97020, 1:5,000, Abcam). AP activity was detected by BCIP/NBT kit (Invitrogen) according to the supplier’s instructions. HRP activity was detected by the Supersignal Western Detection Reagents (Thermo Scientific).
6
Protein Extraction and Immunoblot for Rice Panicle
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Protein extraction and immunoblot assays were performed as modified method below3 (link). Rice young panicle samples were harvested and soluble proteins were extracted with a Plant Total Protein Extraction Kit (Sigma-Aldrich, PE0230). Briefly, pooled 1 mm to 3 mm young panicles were ground to a fine power in liquid nitrogen, then the powder was rinsed with methanol followed by acetone. The supernatant was removed from each sample, and the remaining tissue was then dried and dissolved. Proteins were denatured by adding concentrated SDS loading buffer and boiling for 5 min, then were separated on a precast 4-12% Bis-Tris gel (Tanon, 1808008H). Endogenous OsMPK6 levels were assayed via immunoblot using anti-OsMPK6 antibody at 1:3000 dilution (Sigma-Aldrich, A7104). Phosphorylated OsMPK6 was visualized using an optimized immunoblot analysis with immunoreaction enhancer solutions (TOYOBO, NKB-101) and anti-Phos-OsMPK6 antibody at 1:1000 dilution (CST, 4370), which specifically recognizes the conserved dual-phosphorylated T-E-Y motif of phosphorylated OsMPK6. The loading control was probed using anti-Actin antibody at 1:5000 dilution. (Abmart, M20009)
7
Peptide Antibody Production and Validation
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A specific peptide (N′-CGHSERRHVIGEKDE) was synthesized, and a rabbit polyclonal antiserum was produced and purified by GenScript Corporation (Nanjing, China). The specificity of the anti-pdTPI antibody was verified as shown in Supplemental Figure 2B. The anti-CAT2 polyclonal antibody was prepared and characterized as previously reported (Hu et al., 2010; Yuan et al., 2017) . The anti-pdTPI and anti-CAT2 were used at a working dilution of 1 μg ml -1 . Anti-actin (1:1,000 dilution) (M20009, Abmart), anti-cpHSC70 (1:1,000 dilution) (AS08348, Agrisera), and anti-GAPC (1:1,000 dilution) (AS152894, Agrisera) were used according to the manufacturer's instructions.
8
Protein Extraction and Immunoblot Analysis
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Protein extraction and immunoblot assays were performed as described previously (Zhu et al., 2018) . Approximately 100 mg of shoot apex from 35-day-old seedlings were harvested and ground into a fine powder in liquid nitrogen. Total proteins were extracted using 2 ml immunoprecipitation buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM NaF, 1.5 mM Na 3 VO 4 , 10 mM beta-glycerophosphate, and 13 complete protease inhibitor cocktail [Roche] ). The samples were incubated at 4 C for 2 h with rocking and centrifuged at 13 000 rpm at 4 C for 30 min, and the supernatant was collected. The total protein (1.5 mg) was immunoprecipitated using 50 ml anti-OsFD1 antibody (1:40 dilution; ABclonal Biotechnology) and 50 ml of Dynabeads Protein G (1:40 dilution; Invitrogen). Actin was used as an internal control, with 1 mg total protein extract used to measure actin protein levels by immunoblotting analysis with anti-actin (Abmart M20009, 1:4000 dilution) antibody. The endogenous protein levels of OsFD1 were visualized by immunoblot analysis using anti-OsFD1 (ABclonal). The immunoprecipitated proteins were then detected via immunoblot analysis with anti-OsFD1 antibody (ABclonal Biotechnology; 1:2000 dilution) and anti-phosphoserine antibody (Sigma PSR-45; 1:2000 dilution).
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