Antibodies against phosphorylated (p)-eIF2α (Ser51) (ab32157), p-PKR (Thr451) (ab81303), and total PKR (ab32052) were purchased from Abcam (Cambridge, MA, USA). The antibody against total eIF2α (sc-11386) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The monoclonal antibody clone 16F8 against p-PERK (Thr980) (#3179) was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against PERK (20582-1-AP), GADD34 (10449-1-AP), and β-actin (20536-1-AP) were obtained from Proteintech Group (Chicago, IL, USA). The antibody against PRV (PA1-081) was obtained from Invitrogen (Carlsbad, CA, USA), the monoclonal antibody clone 12D10 against puromycin (MABE343) was obtained from Sigma–Aldrich (St. Louis, MO, USA), and the HRP-conjugated IgG secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Thapsigargin (Tg) (67526-95-8), salubrinal (324895), and puromycin (540222) were purchased from Sigma–Aldrich. The PP1/PP2A inhibitor okadaic acid (OA) (GC16958) was purchased from GlpBio (Montclair, CA, USA). Lipofectamine 2000 was purchased from Invitrogen.
Hrp conjugated igg secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
HRP-conjugated IgG secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of an immunoglobulin G (IgG) antibody that is conjugated with the enzyme horseradish peroxidase (HRP). This conjugated antibody can be used to detect and amplify the signal of a primary antibody that has bound to a target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
β actin 20536 1 ap, by Proteintech (1 mentions)
Ab32157, by Abcam (1 mentions)
Ab32052, by Abcam (1 mentions)
Ab81303, by Abcam (1 mentions)
Sc 11386, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
Gapdh 6c5, by Santa Cruz Biotechnology (1 mentions)
Gfp d5.1, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
C2c12 myoblast, by American Type Culture Collection (1 mentions)
In cell elisa colorimetric detection kit, by Thermo Fisher Scientific (1 mentions)
Screen well fda approved drug library v2, by Enzo Life Sciences (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab50151, by Abcam (1 mentions)
Ab9132, by Abcam (1 mentions)
6 protocols using hrp conjugated igg secondary antibody
1
Antibody-based Analysis of Stress Signaling
2
Western Blot Analysis of STING and LC3B
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Cells were lysed in Cold Spring Harbor NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, and 1.0% Nonidet P-40) containing protease and phosphatase inhibitors (PMSF, NaF, Na3VO4, and Roche PhosSTOP). Lysates were cleared by centrifuging at 10,000 g for 10 min at 4° C, size separated on SDS-PAGE gels, and wet transferred onto polyvinylidene fluoride membrane. Membranes were blocked in tris-buffered saline with 0.05% Tween 20 (TBS-T) and 5% nonfat dry milk for 1 h at room temperature, followed by overnight incubation at 4° C with primary antibodies diluted in TBS-T with 5% BSA. Membranes were washed three times with TBS-T for 10 min, incubated with HRP-conjugated IgG secondary antibody (Jackson Immunoresearch) for 1 h at room temperature, washed three times with TBS-T for 10 min followed once with TBS for 10 min. Lastly bands were visualized with SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and Bio-Rad’s ChemiDoc MP imager. Antibodies to the following were purchased from Cell Signaling Technology: LC3B (D11), Phospho-STING (D8F4W); and Santa Cruz Biotechnology: GAPDH (6C5).
3
Western Blot Analysis of STING Pathway
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Cells were lysed in Cold Spring Harbor NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, and 1.0% Nonidet-P40) supplemented with protease and phosphatase inhibitors (PMSF, NaF, Na3VO4, and Roche PhosSTOP) and then centrifuged at 12,000 g for 15 min at 4°C to get cellular lysate. Equal amounts of protein were loaded and size separated on an SDS–PAGE gel and wet transferred onto polyvinylidene fluoride membrane. The membrane was blocked in tris-buffered saline and Tween 20 (TBS-T) buffer with 5% milk for 1 h at room temperature, followed by overnight incubation with primary antibodies diluted in TBS-T with 5% BSA. Membrane was washed three times with TBS-T buffer for 10 min, incubated at room temperature with HRP-conjugated IgG secondary antibody (Jackson Immunoresearch), washed three times with TBS-T for 10 min followed once with TBS buffer for 10 min, and then developed with SuperSignal West Femto Chemiluminescent Substrate (Life Technologies).
Rabbit antibodies against TBK1 (D1B4), pTBK1 (Ser172, D52C2), STING (D2P2F), p-STING (Ser365, D8F4W), p-STING (Ser366, D7C3S), FLAG (D6W5B), HA (C29F4), and GFP (D5.1) were from Cell Signaling Technology. Rabbit antibody against SURF4 was from Novus. Mouse antibodies against GAPDH and β-Tubulin were from Santa Cruz Biotechnology. Mouse antibody against GM130 was from BD Biosciences.
Rabbit antibodies against TBK1 (D1B4), pTBK1 (Ser172, D52C2), STING (D2P2F), p-STING (Ser365, D8F4W), p-STING (Ser366, D7C3S), FLAG (D6W5B), HA (C29F4), and GFP (D5.1) were from Cell Signaling Technology. Rabbit antibody against SURF4 was from Novus. Mouse antibodies against GAPDH and β-Tubulin were from Santa Cruz Biotechnology. Mouse antibody against GM130 was from BD Biosciences.
4
Western Blot Analysis of Apoptosis Markers
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Cells were harvested and lysed with RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, Tris pH 8.0). Protease inhibitors (Beyotime) were also added. Then 30 µg cell lysates were loaded and separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE, 10%) and subsequently blotted on polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat milk for 1 hour, the membrane was incubated overnight at 4℃ with the following primary antibodies: caspase3 and cleaved caspase3 (1:1,000 dilution, CST, USA); cleaved PARP (1:1,000 dilution, CST); SIVA (1:500 dilution, CST); p53 (1:1,000 dilution, CST); mTOR (1:1,000 dilution, CST); phospho-mTOR (1:500 dilution, CST, USA); S6K (1:1000 dilution, CST, USA); phospho-S6K (1:500 dilution, CST, USA); 4EBP1 (1:1000 dilution, CST, USA) phospho-4EBP1 (1:1500 dilution, CST, USA) ; GAPDH (1:1000 dilution, CST, USA). Then proper HRP-conjugated IgG secondary antibodies (Jackson ImmunoResearch, USA) were used to mark primary antibodies . Finally, the immunoreactive bands were visualized by immobilon Western chemiluminescent HRP substrates (Merck, Millipore, USA).
5
High-throughput drug screening using ELISA
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An ELISA-based high-throughput limited drug screen was designed using an In-Cell ELISA Colorimetric Detection Kit (Thermo Fisher Scientific, Massachusetts, USA). For this, a total of 262 FDA-approved drugs were aliquoted in 384-well microplates derived from the Screenwell FDA-approved drug library V2 (Enzo Life Sciences—Cederlane—Ontario, Canada). Each drug dose from the initial drug screen was based on doses used in the clinic by patients as listed in the Drug Bank database66 (link). C2C12 myoblasts (American Type Culture Collection, Virginia, US, CRL-1772TM) were grown in each well and treated with these drugs or vehicle control for 24 h. Following treatment, antibodies targeting eEF1A2 (1:1000, provided by Dr. Abbott) or utrophin A (1:500; Novocastra, Leica biosystems, Concord, ON, Canada, NCL-DRP2) and HRP-conjugated IgG secondary antibodies (Jackson Immuno Research, Bar Harbor, USA, 111-035-033 and AP124P) were used to detect protein expression levels. Absorbance levels were determined with a Synergy H1 microplate reader. Note that the absorbance levels are standardized to total cell number by using a whole-cell stain in order to control for variation in cell proliferation.
6
Western Blot Analysis of Skeletal Muscle
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C2C12 skeletal muscle cells or mouse muscle tissues were homogenized in urea buffer supplemented with protease inhibitor (Roche). A total of 5–20 μg of protein extracts were resolved on either a 7% SDS-PAGE gel for utrophin A and eEF1A2 analyses, or 10% SDS PAGE gels for CAT and β-GAL analyses. Proteins were transferred overnight at 4 °C onto nitrocellulose membrane (Bio-Rad, Mississauga, ON, Canada, 0.45 μm). Membranes were subsequently washed 4 times with 1× PBS-T (1xPBS, 0.2% Tween) and blocked for 1 h with a 5% skim milk in PBS-T solution. Membranes were incubated with primary antibodies directed against utrophin A (1:500; Novocastra, NCL-DRP2), eEF1A2 (1:1000, provided by Dr. Abbott and Abcam, Toronto, ON, Canada), myc tag (1:1000, Abcam, ab9132), CAT (1:1000, Abcam, ab50151), β-GAL (1:1000, Abcam, ab616), LC3A/B (1:1000; Cell Signaling, Danvers, MA, USA, 12741S) and β-actin (1:10,000; Santa Cruz, sc-47778). Blots were probed with appropriate HRP-conjugated IgG secondary antibodies (Jackson ImmunoResearch, 111-035-033, AP124P and 705-035-003). Protein detection was performed by using ECL reagent (Perkin Elmer, Waltham, MA, USA). The films were quantified using ImageJ (NIH version 1.0) and/or Image Lab. Uncropped scans are available in the Source Data file.
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