We extracted total proteins with grinding buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 1x protease inhibitor), mixed with a 6x SDS sample buffer, and heated in boiling water for 9 min before loading on 8% or 10% SDS-PAGE mini-gels. Gels were transferred to the PVDF membrane (Millipore, Billerica, MA) using an electrotransfer unit (Hoefer Pharmacia Biotech, USA). The blots were washed with PBST washing buffer (phosphate buffer and 0.1% Tween-20) three times for 5 min each, soaked in blocking solution (5% non-fat milk) for 5 min, and then probed with specific primary antibodies such as FIN219 and PHYA monoclonal antibodies [6; this study] and COP1 polyclonal antibodies [45 (link)] as well as PIF3 (AS163954, Agrisera)/4 (AS163955, Agrisera) polyclonal antibodies at 4°C overnight. The membranes were washed with PBST washing buffer twice and then incubated with an appropriate secondary antibody conjugated with horseradish peroxidase for 1 h. The signals were developed using the ECL system (T-pro LumiFast Plus ECL Kit) and detected using LAS-3000 (FUJIFILM).
As163955
Manufactured by Agrisera
The AS163955 is a laboratory equipment product from Agrisera. It is designed to serve a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using as163955
1
Western Blot Protein Detection Protocol
2
Quantification of Gene Expression and Protein Levels
3
Quantifying Tomato Leaf Proteins
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Total proteins were extracted from tomato leaves by homogenization in extraction buffer as described previously (Wang et al., 2019 ). Protein concentrations were measured using Coomassie stain (Bradford, 1976 ). Equal amounts of total proteins from each sample were subjected to 15% SDS‐PAGE and electrotransferred to nitrocellulose membranes (Bio‐Rad, Hercules, CA). The proteins were blotted with antibodies against PIF4 (AS163955; Agrisera) or anti‐HA (Cat. No. 26183; Pierce) and subsequently with horseradish‐peroxidase‐conjugated secondary antibody (anti‐goat, Invitrogen, Sweden). The signals were visualized with enhanced chemical luminescence (ECL).
4
Quantification of Gene Expression and Protein Levels
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Total RNA was isolated from seven days old seedlings grown at the different temperatures by TRIzol method and incubated with DNase (Promega) for 30min at 37°C to remove residual genomic DNA before reverse transcription. 1 μg of total RNA was used for reverse transcription by M-MLV reverse transcriptase (Invitrogen). The transcript levels of target genes were determined by real-time PCR using specific primer sets (Supplementary Table 1 ) and normalized with that of PP2A. Two biological replicates were performed, with four technical replicates each. For protein analysis, seedlings were frozen in liquid nitrogen and homogenized. Total protein was extracted with urea-denaturing buffer (100 mM NaH2PO4, 10 mM Tris-Cl, and 8 M urea, pH 8.0, 1mM PMSF, Protease inhibitor cocktails) and the debris was removed by centrifugation. The extracted proteins were further denatured by boiling at 100°C for 5 min with 1X SDS sample buffer. The protein levels were detected by immunoblot analysis using an anti-myc antibody (Santa Cruz, c-myc (9E10) X antibody, sc-40 X, 1:10000 dilution for western blot), anti-PIF4 antibody (Agrisera, AS16 3955, 1:1000 dilution for western blot), anti-tubulin antibody (Sigma-Aldrich, anti-α-Tubulin antibody, T5168, 1:10000 dilution for western blot), anti-RPT5 (Enzo Life Sciences, BML-PW8770-0025, 1:10000 dilution for western blot).
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