Total protein extracts were prepared using cell lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, 1% Triton X-100, 1% SDS, 5% Glycerol), and equal amounts of protein were separated on 4–15% Mini-PROTECAN TGX Precast Gels (Bio-Rad). The proteins were transferred onto a polyvinylidene fluoride membrane (Millipore) and blocked for 30 min at 25 °C with 3% BSA/Tris-buffered saline solution that contained 0.1% Triton X-100. Thereafter, the membrane was incubated with a 1:300 dilution of anti-ICP0 mouse monoclonal antibody (Abcam, ab6513), 1:5,000 dilution of anti-ICP4 mouse monoclonal antibody (American Type Culture Collection, hb-8183), and 1:5,000 dilution of anti-GAPDH mouse monoclonal antibody (Millipore, MAB374) for 1 h at 25 °C. All first antibodies were diluted with IMMUNO SHOT reagent 1 (Cosmobio). Thereafter, the membrane was incubated with a 1:5000 dilution of anti-mouse immunoglobulins goat polyclonal antibody conjugated with horseradish peroxidase (HRP) (Dako, P0447) for 1 h at 25 °C. The secondary antibody was diluted with IMMUNO SHOT reagent 2 (Cosmobio). Lastly, the proteins were visualized using Immobilon Forte Western HRP substrate (Merck) and chemiluminescence signal was detected with a Luminescent Image Analyzer LAS-4000 mini (Fujifilm).
Immunoshot reagent 1
Manufactured by Cosmo Bio
Sourced in Japan
Immunoshot Reagent 1 is a laboratory reagent used in immunological assays. It is a key component in the detection and quantification of specific proteins, antibodies, or other biomolecules in a sample. The core function of this reagent is to facilitate the binding and visualization of target analytes during the immunoassay process.
Automatically generated - may contain errors
Lab products found in correlation
Immuno shot reagent 2, by Cosmo Bio (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Trueblot anti rabbit igg hrp, by Rockland Immunochemicals (2 mentions)
β actin, by Merck Group (2 mentions)
Ab6513, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Las 4000 mini luminescent image analyzer, by Fujifilm (1 mentions)
Snrnp200, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Hnrnp a1 d21h11, by Cell Signaling Technology (1 mentions)
Hnrnp u, by Abcam (1 mentions)
Nucleolin, by Abcam (1 mentions)
Flag dykddddk tag, by Fujifilm (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
Mouse anti ha, by Merck Group (1 mentions)
Anti gst, by Thermo Fisher Scientific (1 mentions)
7 protocols using immunoshot reagent 1
1
Western Blot Analysis of Herpes Virus Proteins
2
Western Blot Validation of Protein Targets
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Western blotting was performed as previously described [29 (link)]. To confirm the proteins identified by LC-MS/MS, RNA immunoprecipitation samples were transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Chalfont St. Giles, UK) after SDS-PAGE. In other cases, lysate samples were separated by 10–20% gradient SDS-PAGE followed by electrical transfer to PVDF membranes. After blocking with 5% dry milk, membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo Bio, Tokyo, Japan) overnight at 4° C. Horseradish peroxidase (HRP)-conjugated corresponding secondary antibodies (GE Healthcare) were subsequently used. Trueblot anti-rabbit IgG HRP (1:1000; Rockland, Limerick, PA, USA) was used as the secondary antibody to avoid interfering with the immunoprecipitated immunoglobulin heavy and light chains. Bound antibodies were detected using Immunostar LD reagents (Wako). The following antibodies were used: HBV PreS2 (#ab30923, 1:1000), DNA-PK (#ab32566, 1:1000), DHX9 (#ab26271, 1:1000), ILF3 (#ab92355, 1:1000), and snRNP200 (#ab118713, 1:1000) from Abcam (Cambridge, UK); leucine-rich PPR motif-containing protein (LRPPRC; #ARP41093, 1:1000) from AVIVA Systems Biology (San Diego, CA, USA); and β-actin (#5125, 1:2000) from Cell Signaling Technology (Danvers, MA, USA).
3
Western Blotting Procedure for Protein Analysis
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Western blotting was performed as previously described69 (link). Briefly, lysate samples were separated on 5–20% gradient polyacrylamide gel by SDS–PAGE following electrical transfer to PVDF membranes (GE Healthcare). After blocking with 5% dry milk, membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo bio, Tokyo, Japan) overnight at 4 °C. HRP-conjugated corresponding secondary antibodies (GE Healthcare) were subsequently used. Trueblot anti-rabbit IgG HRP (Rockland, Limerick, PA, USA, 1:1,000) was used as the secondary antibody to avoid interfering immunoprecipitated immunoglobulin heavy and light chains. Bound antibodies were detected using Immunostar LD reagents (Wako). The following antibodies were used: Hnrnp-U (#ab180952, 1:1,000) and Nucleolin (#ab22758, 1:1,000) from Abcam (Cambridge, UK); HA tag (#561, 1:10,000) and Syncrip (#RN046PW, 1:1,000) from MBL; Hnrnp-A2/B1 (#R4653, 1:1,000) and β-actin (#A1978, 1:2,000) from Sigma-Aldrich; and YBX1 (D299, 1:1,000), Igf2BP1 (D33A2, 1:1,000) and HnrnpA1 (D21H11, 1:1,000) from Cell Signaling Technology (CST, Danvers, MA, USA).
4
Western Blot Protocol for Protein Analysis
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Western blotting was performed as previously described.33 (link) Briefly, lysate samples were separated on a 10%–20% gradient polyacrylamide gel by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis following electrical transfer to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA). After blocking with 5% dry milk, membranes were probed overnight at 4°C with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo Bio Co, Tokyo, Japan). Horseradish peroxidase–conjugated corresponding secondary antibodies (GE Healthcare, Little Chalfont, United Kingdom) were subsequently applied. Bound antibodies were detected using Immunostar LD reagents (Wako Pure Chemical Industries, Osaka, Japan). The following antibodies were used: Smc5 (#ab18038, 1:10,000 [Abcam, Cambridge, United Kingdom]), β-actin (#A1978, 1:10,000 [Sigma-Aldrich, St. Louis, MO] or #5125, 1:10,000 [Cell Signaling Technology, Danvers, MA]), Hepatitis B preS2 (#NB100-93580, 1:1000 [Novus Biologicals, Littleton, CO]), DDB1 and GST tag ((#5428, 1:1000 and #2625S, 1:1000, respectively [Cell Signaling Technology]), and Flag (DYKDDDDK) tag (#018-22381, 1:1000 [Wako Pure Chemical Industries]). Protein band intensities were quantified through densitometric analysis using ImageJ software version 1.49v (National Institutes of Health, Bethesda, MD).
5
Western Blot Antibody Optimization
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Western blotting was performed as described (Zhang et al. 2019a (link)) with the following primary antibodies: anti-SF3B1 (1:1000; Bethyl Laboratories A300-996A), anti-GST (1:1500; Invitrogen A5800), anti-SUGP1 (1:1000; Bethyl Laboratories A304-675A-M), anti-DDX46 (1:1000; Bethyl Laboratories A301-052A-T) used with Immuno Shot reagent 1 (Cosmo Bio), anti-HA rabbit (1:1000; Abm G166), anti-HA mouse (1:1000; Sigma-Aldrich H3663), and anti-ACTIN (1:2000; Sigma-Aldrich A2066).
6
Western Blot Detection Protocol
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Western blotting was performed as previously described (29 ). Briefly, total lysate samples were separated in 10 to 20% gradient polyacrylamide gels (Fujifilm Wako Pure Chemicals) and transferred to immobilon-P membranes, (polyvinylidene fluoride; Merck). After blocking with 5% dry milk, the membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo Bio) for 16 h at 4 °C. The corresponding horseradish peroxidase (HRP)–conjugated secondary antibodies (GE HealthCare) were then added. Bound antibodies were detected using the Immunostar LD Reagent (Wako Pure Chemical Industries). The antibodies used in this study are listed in Table S4 .
7
Arsenic-induced FOXO protein localization
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Cells were seeded on coverslips in 6-well plates at a density of 3 × 10 5 cells/well. Following the incubation of cells with 10 ppm arsenic acid for 16 hr, cells were fixed and permeabilized using Image-IT ® Fix-Perm kit, following the manufacturer's protocol. After permeabilization, cells were washed with Dulbecco's PBS and non-specific binding sites were blocked in 3% BSA in Dulbecco's PBS. The coverslips were incubated overnight with anti-FOXO1 or anti-FOXO3a antibodies in Immuno Shot Reagent 1 (Cosmo Bio Co., Ltd., Tokyo, Japan) at 4°C. Cells were subsequently incubated with goat-anti-rabbit conjugated Alexa Flour ® 532 (Life Technologies, Carlsbad, CA, USA) in Immuno Shot Reagent 2 (Cosmo Bio Co., Ltd.) for 1 hr. The nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies). Finally, coverslips were mounted and examined under confocal microscope (TCS SP8, Leica, Wetzlar, Germany). Images were captured at × 63 magnification.
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