Western blots were performed as described63 (link). Proteins from 14-day-old seedlings were extracted, resolved in 12% (v/v) SDS-polyacrylamide gels, and transferred to Hybond C-Extra membranes (Amersham Biosciences). The membranes were blocked with 5% (w/v) non-fat milk in Tris buffered saline tween (TBST) buffer and then probed with specific antibodies. Antibodies used included anti-GAPDH (Santa Cruz Biotechnology, sc-365062, dilution, 1:1000), anti-SUL (dilution, 1:1000)35 (link), anti-HYL1 (Agrisera, AS06136, dilution, 1:2000), anti-AGO1 (Agrisera, AS09527, dilution, 1:2000), anti-SE (Agrisera, AS09532A, dilution, 1:1000), anti-H3 (Agrisera, AS10710, dilution 1:3000) and anti-GFP (Abcam, ab290, dilution, 1:3000). After three washes, the membranes were probed with horseradish peroxidase-conjugated, goat-anti-rabbit IgG (Bio-Rad, cat.172-1019) (dilution, 1:2000) or goat anti-mouse IgG (Bio-Rad, cat.170-6516, dilution, 1:2000). The protein signals were detected with the AmershamTM ECLTM Prime Western Blotting Detection Reagent (GE healthcare, RPN2232) and visualized with the Chemiluminescence imaging system (Clinx Science Instruments Co. Ltd., China).
As06136
Manufactured by Agrisera
Sourced in Sweden
AS06136 is a plant-specific antibody used for the detection of proteins in Western blot analysis. The antibody is designed to recognize a specific target protein and can be used to quantify its expression levels in plant samples.
Automatically generated - may contain errors
Lab products found in correlation
As09532a, by Agrisera (6 mentions)
As122102, by Agrisera (3 mentions)
Ab290, by Abcam (2 mentions)
Hybond c extra membrane, by Cytiva (2 mentions)
Amersham ecltm prime western blotting detection reagent, by GE Healthcare (2 mentions)
Sc 365062, by Agrisera (2 mentions)
As09527, by Agrisera (2 mentions)
Goat anti mouse igg, by Bio-Rad (2 mentions)
Goat anti rabbit igg, by Bio-Rad (2 mentions)
A4596, by Merck Group (2 mentions)
A8592, by Merck Group (2 mentions)
Adi spa 818, by Enzo Life Sciences (2 mentions)
As04043, by Abcam (1 mentions)
Gta 20, by Proteintech (1 mentions)
Ab190584, by Abcam (1 mentions)
M4439, by Merck Group (1 mentions)
Gfp trap agarose, by Proteintech (1 mentions)
H6533, by Merck Group (1 mentions)
Anti ha, by Thermo Fisher Scientific (1 mentions)
As09532a, by Abcam (1 mentions)
10 protocols using as06136
1
Western Blot Analysis of Plant Proteins
2
Quantifying Protein Levels in Plant Seedlings
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To determine protein levels, total proteins were extracted from 14-d-old seedlings, resolved in sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS) gels, and then transferred to nitrocellulose membranes. The membranes were blocked with 1× PBS supplemented with 5% (wt/vol) nonfat milk and 0.05% Tween 20 and then incubated with primary antibodies (anti-HYL1, 1:2,000, Agrisera, AS06136; anti-cFBPase, 1:5,000, Agrisera, AS04043; anti-histone H3, 1:3,000, Abcam, ab1791; anti-SE, 1:2,000, Agrisera, AS09532A; anti-GFP, 1:1,000, Sigma-Aldrich, 11814460001; homemade DCL1, 1:2,000) at 4 °C. After 3 washes with 1× PBS supplemented with 0.05% Tween 20, the membranes were incubated with secondary antibodies and then detected with ECL reagent (Amersham, RPN2236). HYL1 phospho-isoforms were detected using phos-tag SDS-PAGE (Fujifilm Wako Pure Chemical Corporation, 190–16721) following the manufacturer’s instructions. Briefly, total proteins were separated on a phos-tag SDS-PAGE gel, transferred onto a PVDF membrane using the wet-tank transfer method, and then probed with the HYL1 antibody.
3
Investigating JANUS Interactions with DCL1 and SE
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To examine the interaction of JANUS with DCL1 and SE, JANUS-FLAG was transiently co-expressed with GFP-DCL1 or MYC-SE in N. benthamiana as described (12 (link)). The expression of these transgenes was directed by the 35S promoter. Total proteins of infiltrated leaves were extracted with an extraction buffer (50 mM Tris–HCl 8.0, 150 mM NaCl, 5% glycerol, 5% Triton X-100, 1 mM EDTA, 1× complete protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride). Immunoprecipitation (IP) was performed on protein extracts using anti-GFP (GTA-20, Chromotek) or anti-FLAG antibodies (A4596, Sigma) coupled to protein G agarose beads. After IP, proteins were separated on a 10% SDS-PAGE and detected with western blot using monoclonal antibodies against GFP (902602, Biolegend) or FLAG (A8592, Sigma). DCL1, SE and HYL1 proteins in Arabidopsis were detected with antibodies against DCL1 (Agrisera, AS122102), SE (Agrisera, AS09532A) and HYL1 (Agrisera, AS06136).
4
Co-Immunoprecipitation Assay for Protein Interactions
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The Co-IP assay was performed as previously described66 (link). To examine the interactions of CWC15 with HYL1, SE, PAB1 and PAG1, MYC-CWC15 was transiently co-expressed with HYL1-2HA, SE-2HA, PAB1-2HA and PAG1-2HA in N. benthamiana leaves, respectively. The interaction between CWC15 and PRP4KA was performed by co-expressing 2HA-CWC15 and FLAG-PRP4KA in N. benthamiana. IP was performed on protein extracts using anti-HA (26181, Thermo Scientific), anti-MYC (GTA020, Bulldog Bio) or anti-FLAG (A4596, Sigma) coupled to protein G agarose beads. After IP, proteins were detected with Western blot using antibodies against MYC (1:5000 dilutions, rabbit monoclonal, M4439, Sigma), FLAG (1:10000 dilutions, mouse monoclonal, A8592, Sigma) or HA (1:5000 dilutions, mouse monoclonal, H6533, Sigma). For the interaction of CWC15 and SE or HYL1 in Arabidopsis, IP was performed with ChromoTek GFP-Trap® Agarose in pCWC15::eGFP-CWC15 transgenic plant. After IP, proteins were detected with Western blot using anti-GFP (1:1000 dilutions, rabbit polyclonal, ab190584, Abcam), anti-HYL1 (1:5000 dilutions, rabbit polyclonal, AS06136, Agrisera) or anti-SE (1:5000 dilutions, rabbit polyclonal, AS09532A, Agrisera) antibodies.
5
Arabidopsis Inflorescence Protein Analysis
6
Arabidopsis Small RNA Biogenesis Protocol
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Arabidopsis thaliana Columbia (Col-0) is wild type. Seeds of dbr1-3 (SALK_047099), hyl1-2 (SALK_064863), and MIR390b::GUS (CS66477) were obtained from the Arabidopsis Biological Resources Center (ABRC, www.arabidopsis.org ). 35S::DCL1-YFP seeds were generated in our laboratory. pHYL1::YFP-HYL1 seeds were obtained from Yuda Fang’s lab. Wild-type and dbr1 fission yeast strains were purchased from Bioneer (http://pombe.bioneer.co.kr ). Anti-DCL1 (Agrisera, #AS122102), anti-HYL1 (Agrisera, #AS06136), anti-SE (Agrisera, #AS09532), anti-Hsc70 (Stressgen, SPA-018), anti-GFP (Covance, #MMS-118R), and anti-FLAG (Sigma, #F7425) antibodies were purchased. Anti-DBR1, anti-NRPB2, and anti-AGO1 antibodies were generated by our laboratory.
7
Protease Sensitivity Assay of AtHYL1
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The protease sensitivity assay using bacterially purified AtHYL1FL and AtHYL1N terminal was performed according to previous reports [35 (link),36 (link)]. The samples were loaded on 12–15% SDS-PAGE, and the protein bands were visualized using Coomassie Brilliant Blue (CBB) staining. The Western blotting of the same experiment setup was further used to analyze by anti-AtHYL1 antibody (5:10,000) (AS06136, Agrisera, Vannas, Sweden), according to the report described earlier [37 (link)].
8
Arabidopsis Inflorescence Protein Analysis
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Protein extracts from inflorescences of Arabidopsis were separated on a 10% Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected with antibodies against HYL1 (Agrisera, AS06136), SE (Agrisera, AS09532A), DCL1 (Agrisera, AS122102), AGO1 (Agrisera, AS09527) and HSC70 (Enzo, ADI-SPA-818).
9
Western Blot Analysis of Plant Proteins
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Western blots were performed as described 65 . Proteins from 14-day-old seedlings were extracted, resolved in 12% (v/v) SDS-polyacrylamide gels, and transferred to Hybond C-Extra membranes (Amersham Biosciences). The membranes were blocked with 5% (w/v) non-fat milk in Tris buffered saline Tween (TBST) buffer and then probed with speci c antibodies. Antibodies used included anti-GAPDH (Santa Cruz Biotechnology, sc-365062, dilution, 1:1000), anti-SUL (dilution, 1:1000) 36 , anti-HYL1 (Agrisera, AS06136, dilution, 1:2000), anti-AGO1 (Agrisera, AS09527, dilution, 1:2000), anti-SE (Agrisera, AS09532A, dilution, 1:1000), and anti-GFP (Abcam, ab290, dilution, 1:3000). After three washes, the membranes were probed with horseradish peroxidase-conjugated, goat-anti-rabbit IgG (Bio-Rad, cat.172-1019) (dilution, 1:2000) or goat-anti-mouse IgG (Bio-Rad, cat.170-6516, dilution, 1:2000). The protein signals were detected with the Amersham TM ECL TM Prime Western Blotting Detection Reagent (GE healthcare, RPN2232) and visualized with the Chemiluminescence imaging system (Clinx Science Instruments Co. Ltd, China).
10
Arabidopsis Protein Profiling by Western Blot
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Thirty micrograms of A. thaliana whole leaf extract (extraction buffer: 100 mM Tris, 10% glycerol, 5 mM EDTA, 5 mM EGTA, 0.15 M NaCl, 0.75% Triton X-100, 0.05% SDS, and 1 mM DTT) was resolved in a 10% denaturing gel, transferred to PVDF membranes and detected by Western blotting. The following antibodies were used: anti-AtPRP40b (AS14 2785, Agrisera; 1: 10,000), anti-DCL1 (AS19 4307; Agrisera; 1:100), anti-CBP80 (AS09 531; Agrisera: 1: 2000), anti-SE (AS09 532A; Agrisera; 1:2000), anti-HYL1 (AS06 136; Agrisera: 1:1000), anti-actin (691001, MP Biomedicals; 1: 1000), anti-rabbit (AS09 602, Agrisera; 1: 20,000), and anti-mouse (sc-2005, Santa Cruz Biotechnology, 1: 10,000).
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