Proteins were separated by SDS-PAGE in 10% gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with PVDF Blocking Reagent for Can Get Signal (Toyobo, Osaka, Japan) for 1 h. The membranes were incubated with primary antibodies against mouse GCAT (1:1,000; sc-86466, Santa Cruz Biotechnology, Dallas, TX, USA), mouse SHMT2 (1:1,000; #12762, Cell Signaling Technology, Danvers, MA, USA), β-ACTIN (1:10,000; A1978, Sigma, St. Louis, MO, USA) or α-TUBULIN (1:50,000; T5168, Sigma, St. Louis, MO, USA) for 1 h at room temperature; Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo) was used for dilution. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies against goat IgG (1:20,000; HAF109, R&D Systems, Minneapolis, MN, USA), rabbit IgG (1:10,000; G-21234, Thermo Fisher Scientific, Waltham, MA,USA) or mouse IgG (1:10,000; G-21040, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature; Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) was used for dilution. Bands were detected with a bio-imaging analyser, EZ-Capture ST (ATTO, Tokyo, Japan) using ECL Select Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).
Can get signal immunoreaction enhancer solution 2
Manufactured by Toyobo
Sourced in Japan, United States
Can Get Signal Immunoreaction Enhancer Solution 2 is a laboratory reagent designed to enhance the signal detection in immunoassays. It is formulated to improve the sensitivity and clarity of immunological reactions.
Automatically generated - may contain errors
Lab products found in correlation
Can get signal immunoreaction enhancer solution 1, by Toyobo (17 mentions)
Triton x 100, by Merck Group (3 mentions)
Trizma base, by Merck Group (3 mentions)
Lumina forte western hrp substrate, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
A1978, by Merck Group (2 mentions)
G 21234, by Thermo Fisher Scientific (2 mentions)
Ecl select western blotting detection reagent, by GE Healthcare (2 mentions)
Nupage novex 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Tween 20, by Merck Group (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Pvdf blocking reagent for can get signal, by Toyobo (2 mentions)
Anti rabbit ig g m hrp conjugate, by Southern Biotech (2 mentions)
Maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Ecl prime western blotting detection reagent, by Cytiva (2 mentions)
For can get signal, by Toyobo (1 mentions)
Haf109, by R&D Systems (1 mentions)
G 21040, by Thermo Fisher Scientific (1 mentions)
22 protocols using can get signal immunoreaction enhancer solution 2
1
Western Blot Analysis of Cellular Proteins
2
Evaluating Cellular Responses to Chemical Compounds
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L-arginine, 2-methoxyestradiol (2-Me), deoxycholic acid, hydrochloric acid, and NaOH (Wako Pure Chemicals, Osaka, Japan); N6-(1-iminoethyl)-L-lysine (L-NIL) (Cayman, Michigan, USA); cell counting kit-8 (Dojindo, Tokyo, Japan); DAF-2DA (Daiichi Pure Chemicals, Tokyo, Japan); hematoporphyrin dihydrochloride (HpD), NaCl, sodium dodecyl sulfate (SDS), Trizma base, Triton X-100, and Tween 20 (Sigma-Aldrich Japan K.K., Tokyo, Japan); NuPAGE Novex 12% Bis-Tris gels (Life Technologies Japan, Tokyo, Japan); polyvinylidene difluoride (PVDF) Blocking Reagent for Can Get Signal, Can Get Signal Immunoreaction Enhancer Solution 1, and Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo, Osaka, Japan); HIF-1α, β-actin, and horseradish peroxidase (HRP)-linked anti-rabbit IgG antibodies (Cell Signaling Technology Japan, K.K., Tokyo, Japan); HCP-1 antibody (Abcam, Cambridge, U.K.); iNOS antibody (BiossAntibodies, Massachusetts, U.S.A.) and Lumina Forte western HRP substrate (EMD Millipore, Billerica, MA, USA) were purchased and used without further purification or modification.
3
Monasucus Pigment Antioxidant Assay
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Monasucus pigment, hypoxantine and malic acid were obtained from Wako Pure Chem. Ind., Ltd. in Japan. Xanthine oxidase (Roche, Basel, Switzerland), 2-[5,5-dimethyl-2-oxo-2λ5-(1,3,2)dioxaphosphinan-2-yl]-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (CYPMPO; Radical Research Inc., Tokyo, Japan), 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Labotec, Tokyo, Japan), β-nicotinamide adenine dinucleotide (NADH), d -glutamic acid, succinic acid and Trizma® base, Triton X-100 and Tween® 20 (Sigma-Aldrich Japan K.K., Tokyo, Japan), Cell Counting Kit-8, MitoSOX and Hoechst 33258 (Dojindo, Japan), deoxycholic acid and hydrochloric acid (Wako Pure Chem. Ind., Ltd., Osaka, Japan), NuPAGE® Novex® 12% Bis-Tris gels (Life Technologies Japan Ltd., Tokyo, Japan), PVDF Blocking Reagent for Can Get Signal®, Can Get Signal® Immunoreaction Enhancer Solution 1 and Can Get Signal® Immunoreaction Enhancer Solution 2 (TOYOBO Co. Ltd., Osaka, Japan), caspase-9 antibody and horseradish peroxidase (HRP) linked anti-rabbit IgG antibody (Cell Signaling Technology Japan, K.K., Tokyo, Japan), ASAH1 antibody (Abcam plc., Cambridge, UK), Lumina forte western HRP substrate (Millipore Co., Billerica, MA), lovastatin (Tokyo chemical industry CO., LTD., Tokyo, Japan), MitoSOX (Life Technologies Inc., Gaithersburg, MD) were purchased and used without further purification or modification.
4
Western Blot Analysis of Nampt
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Cell lysates were separated on SDS-PAGE, transferred onto Immobilon-P membrane (Millipore), and blocked with 10% EzBlock Chemi (ATTO, Tokyo, Japan). The membranes were incubated with rabbit anti-human Nampt polyclonal antibodies (1:3000 dilution, Sigma-Aldrich, V9139) in Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo, Osaka, Japan) for 18 h at 4°C followed by horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibodies (1:10000 dilution, MBL, Nagoya, Japan, #458) in Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) at room temperature for 3 h. The bound antibodies were detected with Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan). Comparable loading of proteins was confirmed using rabbit polyclonal anti β-actin antibodies (1:4000 dilution, MBL, PM053) in Can Get Signal Immunoreaction Enhancer Solution 1.
5
Western Blotting of Cell Lysates
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Neurons were lysed in RIPA Buffer with cOmplete, Mini, EDTA-Free Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche) on ice for 1 hr with regular vortexing to aid lysis. Insoluble proteins were removed via centrifugation, and lysate protein concentration was determined using the Pierce Bicinchoninic Acid Protein Assay Kit (Invitrogen) using a standard curve created with BSA standards of known concentration. Equal quantities of protein (20–50 µg) were resolved on 4–20% gradient SDS-Polyacrylamide gels (Bio-Rad) and then transferred onto Polyvinylidene difluoride membranes (Millipore Sigma). Membranes were blocked in PVDF Blocking Reagent for Can Get Signal (Toyobo) for 1 hr. Primary antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo) and membranes were incubated overnight at 4°C. HRP-labeled secondary antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) and membranes were incubated for 1 hr at room temperature. Blots were developed using Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate (PerkinElmer) and ProSignal ECL Blotting Film (Prometheus Protein Biology Products) according to manufacturer’s instructions. Blots were stripped for reblotting using NewBlot PVDF Stripping Buffer (Licor). Band density was quantified in ImageJ.
6
Western Blot Analysis of HCP-1 Protein
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Hematoporhyrin dihydrochloride (Wako Pure Chem. Ind., Ltd., Osaka, Japan), Cell Counting Kit-8 (DOJINDO LABORATORIES, Kumamoto, Japan), Trizma® base (Sigma-Aldrich Japan K.K., Tokyo, Japan), NaCl (Wako Pure Chem. Ind., Ltd., Osaka, Japan), Triton X-100 (Sigma-Aldrich Japan K.K.) sodium dodecyl sulfate (SDS) (Wako Pure Chem. Ind., Ltd.), deoxycholic acid (Wako Pure Chem. Ind., Ltd.), hydrochloric acid (Wako Pure Chem. Ind., Ltd.), NuPAGE® Novex® 12% Bis-Tris gels (Life Technologies Japan Ltd., Tokyo, Japan), PVDF Blocking Reagent for Can Get Signal® (TOYOBO CO., LTD., Osaka, Japan), Tris Buffered Saline with Tween® 20 (TBST-10X, Cell Signaling Technology Japan, K.K., Tokyo, Japan), HCP-1 antibody (Santa Cruz Biotechnology, Inc., Dallas, TX), Can Get Signal® Immunoreaction Enhancer Solution 1 (TOYOBO CO., LTD.) horseradish peroxidase (HRP) linked anti-rabbit IgG antibody (Cell Signaling Technology Japan, K.K.), Lumina forte western HRP substrate (Millipore Co., Billerica, MA), anti-β-actin (Cell Signaling Technology Japan, K.K.), and Can Get Signal® Immunoreaction Enhancer Solution 2 (TOYOBO CO., LTD.) were purchased and used without further purification or modification.
7
ELISA Screening for Antibody Production
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Unless otherwise stated, mAbs produced in CFPS or E. coli were analyzed by ELISA using our standard protocol described previously. In brief, 50 µL of dead bacterial cells (OD600 nm = 0.1) or 10 µg/mL protein antigens (nontoxic VT2 or influenza vaccine), 0.4% Bovine Serum Albumin (BSA) in PBS as negative control were coated on the MaxiSorp plate (Thermo) overnight, and we followed the standard ELISA protocol using Zipbody as the primary antibody, and anti-rabbit Ig(G + M)-HRP conjugate (Southern Biotech, Birmingham, AL, USA), anti-His tag-HRP conjugate as the secondary antibody, or anti human IgG(H + L) polyclonal antibody-HRP conjugate produced in goat (GeneTex, Irvine, CA, USA). Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) was used for dilution of the secondary antibody.
8
Western Blotting Antibody Incubation
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Western blotting was performed as previously described52 (link). The membranes were incubated with primary antibodies (dilutions ranging from 1:1000 to 1:2000) diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO, Osaka, Japan) overnight at 4 °C. The used HRP-conjugated secondary antibodies (dilutions ranging from 1:2000 for mammalian expression to 1:10,000 for bacterially purified recombinant proteins) were diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) at 25 °C for 1 h.
9
Western Blot Analysis of Protein Lysates
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Cells were washed with phosphate-buffered saline (PBS) and then lysed by sonication in Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing cOmplete protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Aliquots of the cell lysates were mixed with sodium dodecyl sulfate (SDS) sample buffer for 5 min on ice. Polypeptides in the samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 5% to 20% gradient SDS-PAGE gels (Fujifilm Wako Pure Chemical, Osaka, Japan) and electroblotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with SuperBlock (Tris-buffered saline) blocking buffer (Thermo Fisher Scientific), the membranes were incubated overnight at 4°C with primary antibodies diluted in Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan). The membranes were then washed three times with PBS containing 0.05% Tween 20 (PBS-T) and incubated with horseradish peroxidase-conjugated secondary antibody diluted in Can Get Signal immunoreaction enhancer solution 2 (Toyobo) for 2 h at room temperature. After washing, the membranes were treated with SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific), and the chemiluminescent signals on the membranes were detected using a ChemiDoc Touch imaging system (Bio-Rad Laboratories, Hercules, CA).
10
Analyzing Caspase-1 and PRR Proteins
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Caspase-1 in the cell lysate and supernatant and PRR proteins in the cell lysates were analyzed by western blotting. The cells were washed with PBS and lysed in Laemmli sample buffer (Bio-Rad, Hercules, CA) containing β-mercaptoethanol (Katayama Chemical Industries, Osaka, Japan). Proteins were separated by SDS-PAGE through 4–15% polyacrylamide, and transferred onto a nitrocellulose membrane. Membranes were pretreated in Tris-buffered saline-Tween 20 with 5% dry milk for 1 hour, then incubated overnight at 4°C with primary antibodies diluted in the Can Get Signal immunoreaction enhancer solution 1 (Toyobo, Osaka, Japan). Membranes were washed three times with Tris-buffered saline-Tween 20 and incubated with the secondary antibodies diluted in the Can Get Signal immunoreaction enhancer solution 2 (Toyobo) for 1 hour at room temperature or overnight at 4°C.
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