Western blots were performed according to Buck et al. (2019) (link). In short, cells were homogenized in a lysis buffer [50-mm Tris–HCl pH 6.8, 2% SDS, and 1× protease inhibitor (cOmplete; Roche)] with a cell homogenizer (Savant Fastprep FP120; Thermo Fisher) and glass beads. Supernatants according to 1 μg of Chl a were separated on 14% LDS–polyacrylamide gels, blotted on nitrocellulose membranes, blocked with 1× Rotiblock (Carl Roth, Karlsruhe, Germany) and incubated with either an anti-Rubisco antibody (AS03037; Agrisera, Vännäs, Sweden) or an anti-Lhcx antibody, each in 1:10,000 dilution [raised by Buck et al. (2019) (link); AS194367; Agrisera] in 1× Rotiblock. The secondary HRP-labeled goat anti-rabbit antibody (AS09602; Agrisera) was either applied in 1:10,000 (Lhcx) or 1:20,000 (Rubisco) dilution in 1× Rotiblock. Detection was performed in an Odyssey FC (LI-COR) with Roti-Lumin Plus (Carl Roth) as substrate.
As09 602
Manufactured by Agrisera
Sourced in Sweden, United States
AS09 602 is a lab equipment product offered by Agrisera. It is a device designed for laboratory use, but without further details on its core function, a concise and unbiased description cannot be provided while maintaining objectivity. The available information is insufficient to give a detailed factual description without potential for interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
As05085, by Agrisera (3 mentions)
Savant fastprep fp120, by Thermo Fisher Scientific (2 mentions)
Odyssey fc, by LI COR (2 mentions)
Roti lumin plus, by Carl Roth (2 mentions)
As09533, by Agrisera (2 mentions)
Sc 2005, by Santa Cruz Biotechnology (2 mentions)
As14 2785, by Agrisera (2 mentions)
As09 531, by Agrisera (2 mentions)
As19 4307, by Agrisera (2 mentions)
As09532a, by Agrisera (2 mentions)
As03 037, by Agrisera (2 mentions)
Amersham protran, by GE Healthcare (2 mentions)
Roti block, by Carl Roth (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
As142766, by Agrisera (1 mentions)
As142819, by Agrisera (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Se260 mighty small 2, by Hoefer (1 mentions)
Te70x, by Hoefer (1 mentions)
Perfection v850 pro scanner, by Epson (1 mentions)
26 protocols using as09 602
1
Western Blot Analysis of Photosynthetic Proteins
2
Glycine-SDS-PAGE Analysis of NPQ Proteins
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To analyse potential changes in polypeptide composition upon NPQ formation/relaxation, glycine–sodium dodecyl sulphate polyacrylamide gel electrophoresis (glycine–SDS-PAGE; Laemmli 1970 (link)) was used, as in Qin et al. (2015b (link)). Stacking and separation gels of 4 and 12% acrylamide/bis-acrylamide (29:1) mix, respectively, were employed. Samples were denatured with a lithium dodecyl sulphate sample buffer (Ikeuchi and Inoue 1988 ) at 40 °C for 10 min, and 10 μg Chl of chloroplasts was loaded per lane. After electrophoresis, gels were used for electroblotting onto nitrocellulose membrane (GE Healthcare, UK) and the proteins were incubated overnight at 4 °C with antibodies raised against PsbS (Agrisera AS09533, 1:2000), LHCSR3 (Agrisera AS142766, 1:1000) and LHCSR1 (Agrisera AS142819, 1:1000). The β subunit of ATP synthase (ATP-B, Agrisera AS05085, 1:2000) was used as loading control. Enhanced chemiluminescence (WSE-6200HLuminiGraph II chemiluminescent imaging system, ATTO, Japan) was used to visualise protein bands after incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Agrisera AS09602, 1:20000).
3
Leaf Protein Extraction and Analysis
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Total leaf proteins were extracted in the sample buffer as described by Mihailova et al. [8 (link)]. The protein content was determined according to Bradford [54 (link)]. Isolated samples were separated on 12% (dehydrins) or 16% (ELIPs) SDS-PAGE (SE260 Mighty Small II, Hoefer, Holliston, MA, USA) according to Laemmli [55 (link)], modified by adding 8.0% glycerol to stacking and separating gels using a constant current of 20 mA per gel. Each lane contains 30 μg total leaf protein. Using semi-dry transfer (TE70X, Hoefer, Holliston, MA, USA) the proteins were blotted on nitrocellulose membrane for 90 min at a current of 1 mA cm−2. Prestained protein standard (Precision Plus Protein™ Dual Color Standards, Bio-Rad, Hercules, CA, USA) was used for monitoring electrophoresis separation and transfer efficiency. Blots were probed with primary antibodies against ELIPs (AS06 147A, Agrisera, Vännäs, Sweden) and dehydrin K-segment (AS07 206A, Agrisera, Vännäs, Sweden). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was used (AS09 602, Agrisera, Vännäs, Sweden). The resulting bands were visualized by chemiluminescence, and signals were recorded on X-ray Blue films (Carestream Dental LLC, Atlanta, GA, USA). Films were scanned using an Epson Perfection V850 PRO scanner (Seiko Epson Corporation, Suwa, Japan).
4
Protein Extraction and Western Blotting
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Plant material was extracted by grinding precooled samples in liquid nitrogen with a pestle to a fine powder44 (link). The extraction buffer (50 mM Tris–HCl, pH 7, and 1% SDS with Sigma P9599 protease inhibitor cocktail) was added, vortexed vigorously, and then centrifuged for 30 min at 14,000 rpm to obtain a supernatant. Protein content in the supernatant was quantified with Bio-Rad DC reagent (Lowry method). Samples were dissolved in Laemmli buffer, treated at 95 °C for 10 min, and loaded (40 mg) onto Invitrogen NuPAGE gels (10%Bis–Tris Midi Gels). After electrophoresis, proteins were transferred to a PVDF membrane using the Bio-Rad Trans-Blot turbo transfer pack. An anti-ADH antibody (Agrisera AS10685, Agrisera, Vännäs, Sweden), an anti-PDC antibody (Agrisera AS10691; Agrisera, Vännäs, Sweden), and secondary goat anti-rabbit IgG HRP conjugated antibody (Agrisera AS09 602; Agrisera, Vännäs, Sweden) were used. Detection was performed using the LiteAblot TurboChemiluminescence substrate (Euroclone). amido black staining was performed to check equal loading. The blot was stained for 10 min [0.1% amido black (Sigma Aldrich, Milan, Italy), 45% methanol, 10%acetic acid] and then washed in a destaining solution (90%methanol/2% acetic acid/8% water) for 2 min.
5
Leaf Protein Extraction and Western Blotting
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Leaf proteins were extracted using extraction buffer (50 mM Tris-HCl pH 7.8) containing 1 mM EDTA, 2% Triton X-100, 10% glycerol, and 0.5 mM of phenylmethylsulfonyl fluoride (PMSF). Aliquots containing 60 μg of protein from a given supernatant preparation were mixed with 4x Laemmli Sample buffer (BIO-RAD, CA, USA), heated at 95 °C for 5 min and separated by electrophoresis on a Mini-PROTEAN Any kD TGX Stain-Free Gel (BIO-RAD, USA) under denaturating conditions. For immunoblot analysis, proteins were transferred to a nitrocellulose blotting membrane by semidry electroblotting, in a Trans-Blot Turbo Transfer System (BIO-RAD, USA). Membranes were blocked overnight at 4 °C, using Western Blocker solution (Sigma, MO, USA). Then, for immunodetection, membranes were incubated overnight at 4 °C with PTOX antibody (1:10,000 dilution, AS16 3692, Agrisera). Detection was performed with HRP-conjugated secondary antibody (1:25,000 anti-rabbit IgG, AS09 602, Agrisera) and ECL detection reagents (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. Both Stain-Free and immunoblots were imaged using the Chemidoc MP system (BIO-RAD, USA).
6
Immunoblotting Analysis of WHY1 Protein
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Total proteins were extracted with protein extraction buffer (Agrisera, Vannas, Sweeden) supplemented with 5 mM DTT and the cocktail of protease inhibitors to prevent protein degradation. An amount of 10 µg of proteins were separated on 15% acrylamide SDS–PAGE and transferred to 0.45 µm nitrocellulose membrane (Amersham, Buckinghamshire, U.K., catalogue number 10600003). All proteins apart from WHY1 were detected with rabbit polyclonal primary antibody (Agrisera) and secondary HRP-linked anti-rabbit (1 : 10000, Agrisera catalogue number AS09 602).
For immunological detection of WHY1, the antibodies were directed toward the synthetic peptide of recombinant HvWhy1 protein (PRQYDWARKQVF) in rabbits and antibodies were affinity-purified (Generon, Slough, U.K.). The specificity of immunodetection was validated using pre-immune sera.
For immunological detection of WHY1, the antibodies were directed toward the synthetic peptide of recombinant HvWhy1 protein (PRQYDWARKQVF) in rabbits and antibodies were affinity-purified (Generon, Slough, U.K.). The specificity of immunodetection was validated using pre-immune sera.
7
Thylakoid Membrane Protein Analysis
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Thylakoid membranes and soluble proteins were extracted from leaves as previously reported (Lepistö et al. 2009 ). Protein content was determined with the Bio‐Rad Protein Assay Kit and chlorophyll (Chl) content according to Porra et al. (1989 ). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blotting were done as described earlier (Nikkanen et al. 2016 ). PVDF membranes were probed with specific antibodies against PsbS (Agrisera, AS09 533), ZE (Agrisera, AS08 289), VDE (Eskling and Åkerlund 1998 ), STN7 (Agrisera, AS16 4098), and phosphothreonine (P‐Thr, New England Biolabs). A horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit secondary antibody (Agrisera, AS09 602) was used for detection of proteins. All blot images are representative of at least three biological replicates. Quantifications of protein content were performed using the ImageJ software (Schneider et al. 2012 ) and normalized according to the intensity of Li‐Cor Revert Total Protein Stain or Coomassie brilliant blue stain. Statistical significance was determined by two‐tailed Student's t‐tests for unequal variances. P < 0.05 was interpreted as statistically significant.
8
Quantitative Western Blot Analysis of Protein Expression
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The primary antibodies used were H+ ATPase (1:1,000; AS07 260) and AtpB (1:10,000; AS05 085) from Agrisera. The secondary antibody used was HRP-conjugated goat anti-rabbit IgG (1:100,000 for quantification of the condition-dependent PMA expression and 1:1500 for detection of the exogenous PMA protein; AS09 602) from Agrisera. The dilution rate of the secondary antibody was adjusted according to the manufacturer protocol of the developing reagents. In quantitative analysis of protein expression levels, the band intensity was measured using ImageJ software. Detailed experimental procedures are supplied in Supplementary Method 4.
9
Western Blot Analysis of Protein Extracts
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Protein extracts for western blot analysis were prepared using buffer containing 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 0.25% NP-40, 1 mM PMSF, 1x protease inhibitor complete EDTA-free (Roche), and 10 μM MG132. Protein extracts were separated by 13% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (PVDF; Millipore), and analyzed by western blotting using antibodies at dilutions recommended by the manufacturer: anti-c-Myc HRP (9E10 cat num# MA1-980-HRP) anti-BG1 (Agrisera, AS20 4419), anti-H3 (Abcam, ab18521), and anti-rabbit (Agrisera, AS09 602).
10
Protein Separation and Western Blot Analysis
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The samples for Western blot analysis were first subjected to SDS-PAGE using 12% acrylamide gel with 6M urea to separate proteins according to size. The samples were loaded based on the Bradford assay to 1–4μg protein per well (see each experiment for details), with 1:1 vol buffer containing 138mM Tris–HCl pH 6.8, 6M Urea, 22.2% glycerol, 4.3% SDS, 10% β-mercaptoethanol, and a small volume of Bromophenol blue. The separated proteins were electro-transferred from the acrylamide gel to Immobilon PVDF membrane (Millipore). The PVDF membrane was blocked with 5% milk (BIO-RAD blotting grade blocker) and incubated with first antibody and second antibody and finally with ECL™ Western blotting Detection Reagent (Amersham™ GE Healthcare) before imaging on film. The protein-specific antibodies used in the assay were α-ADO (custom made, see Generating ADO-specific antibody above) at 1:1000 dilution, α-PsaB (AS10 695, Agrisera) at 1:1000 dilution, and α-ATPase β (AS05 085, Agrisera) at 1:5000 dilution. The secondary antibody (AS09 602, HRP conjugated Goat anti-Rabbit IgG (H&L), Agrisera) was used at 1:10000 dilution.
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