The crude extracts from wild-type and mutant zebrafish embryos were separated by SDS-PAGE, blotted onto an Immobilon membrane, and probed with anti-zebrafish Pou5f3 rabbit antibody (37) or anti-Rpl11 rabbit antibody (Abcam, ab79352). The crude extracts from mouse oocytes and embryos were separated by SDS-PAGE with Bolt 4 to 12% Bis-Tris Plus gels (Novex) and blotted onto the Immobilon membrane using a Bolt Mini Blot Module (Novex). The membranes were probed with anti-hnRNP D (this study), anti-Gemin5 (this study), or anti-Dhx9 mouse antibody (this study).
Bolt mini blot module
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bolt Mini Blot Module is a compact and efficient wet transfer system designed for the quick and reliable transfer of proteins from polyacrylamide gels to membranes. It features a simple and user-friendly design, allowing for the easy assembly and disassembly of the transfer stack. The module is capable of handling mini-format gels and is compatible with a variety of buffer systems.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon fl, by Merck Group (3 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Irdye 800, by Rockland Immunochemicals (3 mentions)
Odyssey scanner, by LI COR (3 mentions)
Alexa fluor 680 dye, by Thermo Fisher Scientific (2 mentions)
Anti cyclin b1, by Abcam (1 mentions)
Anti mad2, by Fortis Life Sciences (1 mentions)
Anti tdrd3, by Cell Signaling Technology (1 mentions)
Anti γ tubulin, by Merck Group (1 mentions)
Anti hur, by Santa Cruz Biotechnology (1 mentions)
Anti dig ap, by Roche (1 mentions)
Anti flag, by Merck Group (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Image studio, by LI COR (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Mini gel tank, by Thermo Fisher Scientific (1 mentions)
8 protocols using bolt mini blot module
1
Western Blot Analysis of Zebrafish and Mouse Embryos
2
Western Blot Analysis of Ovarian Proteins
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(Novex), blotted onto an immobilon membrane using a Bolt Mini Blot Module (Novex), and probed with anti-Emi2 (this study), anti-cyclin B1 (Abcam; V152), anti-γ-tubulin (Sigma; T6557), anti-Tdrd3 (Cell Signaling Technology; D302G) and anti-Mad2 (Bethyl; A300-301A) antibodies. The crude extracts from mouse ovaries and rabbit reticulocyte lysates and the immunoprecipitates were separated by SDS-PAGE, blotted onto an immobilon membrane, and probed with anti-Pum1 (Bethyl; A300-201A), anti-HuR (SANTA CRUZ; sc-5261), anti-ELAVL2 (HuB) (Proteintech; 14008-1-AP), anti-FLAG (Sigma; F1804-1MG), anti-Tdrd3 (Cell Signaling Technology; D302G) and anti-DIG-AP (Roche) antibodies. The intensity of signals was quantified using ImageJ software.
3
Western Blot Analysis of LASV GP Antibodies
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Purified GP fractions were mixed with NuPAGE LDS sample buffer (Thermofisher, Waltham, MA), boiled for 10 minutes and separated on NuPAGE™ 4-12% Bis-Tris Protein Gels (Thermofisher, Waltham, MA). Subsequently, gels were washed in deionized water three times for 5 minutes, stained with SimplyBlue™ SafeStain (Thermofisher, Waltham, MA) for one hour and destained overnight with distilled water. For western blotting, resolved protein from an unstained gel was transferred to nitrocellulose membranes by Bolt Mini Blot Module (Thermofisher, Waltham, MA). The membranes were blocked with TBS containing 4% non-fat milk powder for 1 hour at RT and subsequently probed with a panel of characterized anti-LASV GP human monoclonal antibodies (48 (link)) (provided by the Walter Reed Army Institute of Research) at a 1:1,000 dilution in Tris-buffered saline with 0.2% Tween-20 (v/v) (TBST) (pH 7.2) containing 4% non-fat milk powder overnight at 4°C with gentle agitation. After three 5-minute washes with TBST, the membranes were incubated with IRDye® 800CW goat anti-human IgG secondary antibody (Li-Cor, Lincoln, NE) at a 1:10,000 dilution for another hour. The membranes were washed three times with TBST and imaged using an Odyssey® CLx Imaging System and processed using Image Studio™ (Li-Cor, Lincoln, NE).
4
Western Blot Analysis of Inflammatory Signaling
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The protein samples were separated on precast gradient polyacrylamide gels (Bolt™ 4–12% Bis‐Tris Plus Gels, Thermo Fisher Scientific) and transferred to nitrocellulose membranes (GE Healthcare Life Science, Marlborough, MA, USA) by using Bolt Mini Blot Module and Mini Gel Tank (Thermo Fisher Scientific), according to the manufacturer's recommendations. The membrane was blocked in 5% BSA. The blocked membrane was probed with a primary antibody and HRP‐conjugated secondary antibody. Following a repeat of the wash step, the membrane was kept in enhanced chemiluminescence detection reagents (Thermo Fisher Scientific) for 1 min. Signal intensity was measured with an image analyser (ChemiDoc™ XRS+ system, Bio‐Rad Laboratories). The primary antibodies used were anti‐IL‐1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4998 (Abcam, Cambridge, MA, USA), anti‐IFN‐γ (Abcam), anti‐GAPDH, anti‐ JAK2, anti‐phospho‐JAK2, anti‐STAT 1 and 3, anti‐phospho‐STAT1 and 3, anti‐IKKα, anti‐phospho‐IKKα, anti‐IκBα, anti‐phospho‐IκBα, anti‐NF‐κB and anti‐phospho‐NF‐κB p65 were purchased from Cell Signaling Technology (Beverly, MA, USA). In addition, anti‐Lamin B from Invitrogen was used.
5
Western Blot Analysis of Zika Virus Proteins
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The supernatant of an S2 cell culture collected one week postinduction was mixed with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher, Waltham, MA), boiled for 10 min, and run on NuPAGE 4 to 12% bis-Tris protein gels (Thermo Fisher, Waltham, MA). Protein was transferred to a nitrocellulose membrane with a Bolt miniblot module (Thermo Fisher, Waltham, MA) and blocked with 1% nonfat dry milk (Nestlé United States Inc., Solon, OH) in PBST, pH 7.4, with 0.01% sodium azide for 1 h at room temperature or overnight at 4°C. Membranes were probed with a 1:3,000 dilution of flavivirus E-specific 4G2 antibody (1.6 mg/ml) or convalescent-phase PRVABC59 ZIKV-infected mouse or NHP serum at a dilution of 1:1,000 for 1 h at room temperature. After being washed with PBST, membranes were incubated with alkaline phosphatase-conjugated, goat anti-mouse or anti-human IgG antibody (Southern Biotech, Birmingham, AL) for another hour. The membranes were washed three times with PBST and developed with a nitroblue tetrazolium (NBT)-BCIP (5-bromo-4-chloro-3-indolylphosphate) (Promega, Madison, WI) solid-phase alkaline phosphatase substrate solution.
6
Immunoblotting of Mouse Embryonic Fibroblasts
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MEFs were plated at different densities to reach full confluency at 24, 48, 72 or 96 h post OHT treatment (with the exception of Figure 5E ). Lysates were separated at 80 V on 10% acrylamide gel for 2 h. Transfer was carried out at 20 V for 1.5 h using Bolt Mini Blot Modules (Life Technologies) to Immobilon-FL (Millipore) membranes. After blocking in Odyssey blocking buffer (LI-COR) for 30 min, the membranes were incubated overnight with 1:1000 mouse monoclonal anti-Viperin (MaP.VIP | #MABF106) (Millipore), rabbit anti-mouse p56 (gift from Ganes Sen, Cleveland Clinic, Cleveland, OH, USA), mouse monoclonal anti-β-tubulin (TU-06 | ab7792) (Abcam) or monoclonal rabbit anti-STING (D2P2F | #13647) (Cell Signaling). Conjugated secondary with Alexa Fluor® 680 dye (Life Technologies) or IRdye800 (Rockland) was subsequently used to image the proteins at 700 or 800 nm with an Odyssey scanner (LI-COR).
7
PNPT1 Knockdown and Western Blot
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For PNPT1-RNAi studies, 10,000 or 80,000 MeWo cells per well—96-well plate and 12-well plate, respectively—were reversed-transfected with 10 nM of siRNA-RNAiMax mix (9 µL lipofectamine RNAiMax/µL siRNA at 40 µM) for 16 h, rinsed with complete RPMI, and stimulated with 1000 IU human IFN-β for another 24 h. For western blot analyses of PNPT1, cell lysates were separated at 80 V on 10% acrylamide gel for 2 h. Following transfer at 20 V for 1.5 h using Bolt Mini Blot Modules (Life Technologies) to Immobilon-FL (Millipore) membranes, membranes were blocked 30 min in blocking in Odyssey blocking buffer (LI-COR). The membranes were subsequently incubated overnight with 1:1000 rabbit polyclonal anti-PNPT1 (ab157109, Abcam), or rat monoclonal anti-β-tubulin (ab6160, Abcam). Finally, conjugated secondary antibodies with IRdye800 (Rockland) were used to image the proteins at 800 nm with an Odyssey scanner (LI-COR).
8
Western Blot Analysis of Immune Proteins
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MEFs were seeded in 24-well plates at a density of ∼25 000 cells per well for 48 h and 50 000 cells per well for 24 h. Analyses were carried out as previously described (18 (link)). Cell lysates were separated at 80 V on 10% acrylamide gels for 2 h. Following transfer at 20 V for 1.5 h using Bolt Mini Blot Modules (Life Technologies) to Immobilon-FL (Millipore) membranes, membranes were blocked 30 min in Odyssey blocking buffer (LI-COR). The membranes were subsequently incubated overnight with 1:1000 mouse monoclonal anti-Viperin (MaP.VIP | MABF106, Millipore), rabbit anti-mouse p56 (gift from G. Sen, Cleveland Clinic, Cleveland, OH, USA), rabbit anti-mouse Mda5 (D74E4 | 5321, Cell Signaling Technology) or mouse monoclonal anti-β-tubulin (TU-06 | ab7792, Abcam). Finally, conjugated secondary antibodies with Alexa Fluor® 680 dye (Life Technologies) or IRdye800 (Rockland) were used to image the proteins at 700 or 800 nm with an Odyssey scanner (LI-COR).
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