A western blotting analysis was performed as described previously [26 (link)]. Cells were washed twice with phosphate-buffered saline (PBS) and lysed by incubation in Lysis A buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH7.0), 5 mM EDTA, 50 mM sodium chloride, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, a tablet of Complete Mini (Roche Diagnostics), and a phosphatase inhibitor cocktail (Sigma Aldrich). The proteins were resolved using SDS-PAGE and were transferred to a PVDF membrane (Immobilon; Millipore, Billerica, MA, USA). After blocking with Tris-buffered saline (TBS) containing 0.02% Tween 20 and 5% nonfat milk, the strips of membrane were exposed to anti-KIAA1199 antibody (epitope sequence, IHSDRFDTYRSKKESERLV; ordered from IBL, Fujioka, Japan), anti-phospho-serine antibody (Millipore), or anti-β-actin antibody (Cell Signaling, Beverly, MA, USA). The cells were incubated with HRP-conjugated anti-rabbit IgG antibody, and the proteins were visualized using an ECL Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK).
Anti phosphoserine antibody
Manufactured by Merck Group
Sourced in Japan, United States
The Anti-phosphoserine antibody is a laboratory reagent used to detect and quantify the presence of phosphorylated serine residues in proteins. It is a highly specific and sensitive tool for researchers studying protein post-translational modifications and signal transduction pathways.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Immobilon, by Merck Group (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Complete mini, by Roche (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti α synuclein, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Anti flag, by Merck Group (1 mentions)
Glutathione sepharose 4b resin, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Ecl advance kit, by GE Healthcare (1 mentions)
Peptone, by HiMedia (1 mentions)
7 protocols using anti phosphoserine antibody
1
Western Blot Analysis of KIAA1199
2
Antibody-based Protein Interaction Analysis
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Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and Lipofectamine® LTX with PLUS reagent were purchased from Invitrogen (Carlsbad, CA, USA). Protein A-Sepharose beads were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Anti-c-Myc, anti-tubulin, anti-α-synuclein, and anti-GFP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FLAG and anti-DAPK1 antibodies were purchased from Sigma-Aldrich (St. Luis, MO, USA). Anti- phospho-α-synuclein (S129) antibody was purchased from Abcam (Cambridge, UK). Anti-phosphoserine antibody, peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies were purchased from Millipore (Billerica, MA, USA). Alexa Fluor® 488-conjugated anti-mouse and Alexa Fluor® 594-conjugated anti-rabbit secondary antibodies were purchased from Invitrogen. Enhanced chemiluminescence (ECL) reagent was purchased from Abclon (Seoul, Korea) and Advansta (Menlo Park, CA, USA). All other chemicals used in the study were analytical grade commercial products and purchased from Sigma-Aldrich.
3
Proteomic Analysis of Bacillus subtilis Spore Coat
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Whole cell lysates were prepared from sporulating cultures of B. subtilis and resolved by SDS–PAGE (Seyler et al., 1997 ). σA, CotB, CotG and CotH were immunodetected in spore coat extracts or whole cell lysates using rabbit polyclonal antibodies of established specificity and as previously described (Zilhao et al., 1999 ; 2004 ; Fujita, 2000 ). An anti‐Phosphoserine antibody was obtained from Milipore and used according to the manufacturer's guidelines.
4
Purification and Phosphorylation Analysis of Recombinant Proteins
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Ni-NTA agarose (Qiagen) and Glutathione-Sepharose 4B resin (Thermo Scientific) were used to purify the recombinant proteins for in vitro binding assays. NST1-GST and NST1(S316A)-GST fusion proteins were incubated with or without SnRK2s-6xHis fusion proteins in kinase buffer (50 mM Hepes [pH 7.4], 10 mM MgCl2, 1 mM dithiothreitol, 30 μM ATP) at 30 °C for 2 h. The reaction was stopped and divided into two equal portions. One group of proteins was separated by SDS/PAGE (Thermo-Fisher NuPAGE 4 to 12%, Bis-Tris, 1.5 mm, Mini Protein Gel, 10 well) and stained with Coomassie brilliant blue as the loading control. The other group was also separated by PAGE, and the gel was used for Western blotting with antiphosphoserine antibody (Sigma P5747) to detect phosphorylated proteins.
5
Glucagon-Induced Serine Phosphorylation
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For phosphorylation experiments, HepG2-GCGR cells were transfected with YWHAB and allowed to recover for 48 hours. HepG2-GCGR cells were then pre-incubated in DMEM supplemented with 0.5% fatty acid free BSA for 30 minutes. HepG2-GCGR cells were then stimulated with 100 nM/L glucagon in DMEM supplemented with 0.5% fatty acid free BSA for 15 minutes. Total cell lysate was collected using the same lysis buffer used in affinity purification with the addition of phosphatase inhibitors (10 mM/L sodium orthovanadate and sodium fluoride). Anti-phosphoserine antibody (1:1000 dilution; Sigma-Aldrich, United States) and HRP-conjugated mouse secondary antibody were used for detection of total serine phosphorylation. The membranes were developed with the ECL advance kit (GE Healthcare) and imaged using the Kodak ImageStation4000 Pro (Care stream Health Inc, Rochester, New York).
6
Characterization of Nisin-Resistant Enterococcus faecalis
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Media components, Beef Extract (catalog number: RM002), Peptone (catalog number: CR001), Yeast Extract (catalog number: RM027) and Sodium Chloride (catalog number: TC046) for bacterial culture were purchased from HiMedia Laboratories (Mumbai, India). Anti-phosphoserine antibody was purchased from Sigma-Aldrich Chemical Co., St. Louis, MO (catalog number: SAB5200086-400UL). Inhibitor molecules were purchased from Enamine Ltd., Ukraine. PAGE reagents and buffers were purchased from Biorad and PVDF transfer membrane, 0.2 µm (Catalog number: 88520) was purchased from Thermo Fisher Scientific, Inc. Mini-PROTEAN® Tetra Cell, 2-gel, 10-well combs, 1.0 mm (catalog no. 1658003) from Biorad was used for PAGE and blotting was performed using semi-dry blotting unit (Scie-Plas Ltd., UK). Sensitive and resistant E. faecalis (NisR-147) bacteria were previously isolated from raw buffalo milk in our lab [5 (link), 23 (link)]. The nisin-resistant strain used here was found to be resistant to chloramphenicol, ampicillin, ciprofloxacin, rifampicin, vancomycin, carbenicillin, linezolid, oxacillin, and fosfomycin with minimum inhibitory concentrations (MICs) varying between 4 µg/mL (ciprofloxacin) and 512 µg/mL (fosfomycin) [5 (link)].
7
Characterization of Purified Δ39aa-hMCD
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The integrity of purified Δ39aa-hMCD was determined by Coomassie brilliant blue (CBB)staining and western blotting analyses (Karger et al. 2008; (link)Park et al. 2008b ). Prior to electrophoresis, purified samples were boiled for 5 min at 95°C with protein denaturing buffer (Nacalai Tesque, Kyoto, Japan). Samples were electrophoresed in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel with a Mini-protean system (Bio-Rad, Hercules, CA, USA) at 150 V for 45-60 min in Tris-glycine buffer (25 mM Tris, 250 mM glycine, pH 8.3 and 0.1% SDS). After electrophoresis, the SDS-PAGE gel was stained with Coomassie blue staining solution. For Western blotting analysis, the separated proteins on a SDS-PAGE gel were transferred to PVDF membranes (GE Healthcare) by electroblotting on a semi-dry blotter (Bio-Rad) at 15 V for 1 h. To detect the purified Δ39aa-hMCD and their phosphorylation, a mouse anti-FLAG antibody (Wako Pure Chem. Ind. Ltd., Osaka, Japan), anti-phosphoserine antibody and anti-phosphotyrosine (Sigma-Aldrich) were used as primary antibodies, respectively. An anti-mouse IgG-HRP (GE Healthcare) was used as a secondary antibody in both cases.
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