Each adherent cell line was washed with 1X PBS. Cells were lysed in RIPA buffer (Sigma, catalogue no. R0278), followed with a 30-minute (min) incubation on ice. Whole cell lysate was collected after centrifugation at 20,000 g for 20 min. For each sample, 20 μg of protein was run on a 4–20% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin in TBST (50 mmol/L Tris pH 7.5, 150 mmol/L NaCl, and 0.1% Tween 20), membranes were probed overnight at 4°C with anti-SETD2 (Sigma, catalogue no. HPA042451) and anti-beta-actin (Cell Signaling Technology, catalogue no. 4970) antibodies. After washing with TBST, membranes were probed with either goat anti-mouse IgG-HRP (Promega, catalogue no. W4021) or goat anti-rabbit IgG-HRP (catalogue no. W4011). ImageJ software was used for blot intensity quantification.
Goat anti mouse igg hrp
Manufactured by Promega
Sourced in United States, Germany
Goat anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Promega (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (3 mentions)
Anti setd2, by Merck Group (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Polysorb elisa plates, by Thermo Fisher Scientific (2 mentions)
Compat able protein assay preparation reagent set, by Thermo Fisher Scientific (2 mentions)
Dbcamp, by Merck Group (2 mentions)
Antibodies specific to cav 1 antibody and pten, by Santa Cruz Biotechnology (2 mentions)
Antibody specific to β actin, by Merck Group (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
Goat anti rabbit igg hrp, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mouse anti gapdh clone g 9, by Santa Cruz Biotechnology (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Goat anti rabbit igg hrp, by Agilent Technologies (1 mentions)
14 protocols using goat anti mouse igg hrp
1
SETD2 Protein Expression Analysis in Adherent Cells
2
VLP-ELISA for MnPV Antibody Detection
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VLP-ELISAs were performed as previously described (29 (link)). Briefly, 96well PolySorb ELISA plates (Thermo Fisher Scientific, Rockford, IL, USA) were coated with 100 ng/well purified high quality MnPV L1-VLPs in 50 mM carbonate buffer pH9.6. The next day, plates were blocked with CBB and incubated for 1 h with three-fold serial dilutions (ranging from 1:100 to 1:656,100) of Mastomys sera in CBB. Then, plates were washed four times with PBST and incubated with goat anti-mouse IgG-HRP (1:10,000 in CBB, Promega GmbH, Walldorf, Germany). After four washes, color development and measurement was performed as described for the GST-ELISA. Antibody titer represents the last reciprocal serum dilution above the blank. The cut-off was set to a titer of 300 based on previous experiences with measuring sera of animals from virus-free and naturally MnPV-infected colonies (28 (link)).
3
Western Blotting of Influenza Proteins
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Western blotting was performed using the Thermo scientific miniblot module. SDS-PAGE of reduced samples was run on Bolt Bis-Tris 4–12% premade gels in MOPS. Blotting was performed onto 0.2nm nitrocellulose membranes at 15V for 30 minutes. Blots were blocked with 5% milk in PBS-0.05% Tween 20 for 1h at room temperature, then incubated overnight at 4°C with primary antibodies at 1:2,000: rabbit anti-PA polyclonal (Genetex), mouse anti-β-actin (Sigma, clone AC-74), and mouse anti-influenza NP (Kerafast, clone HT103). Secondary antibodies (1:3,000) goat anti-Rabbit IgG-HRP (Sigma) and goat anti-mouse IgG-HRP (Promega) were incubated with the blots for 1h at room temperature and the blots were developed using Bio-Rad Clarity Western ECL Substrate. Images were taken on the Bio-Rad ChemiDoc MP imaging system.
4
Immunoprecipitation and Western Blotting
5
Protein Analysis of Adipocyte Markers
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For protein analysis, cells were harvested in SDS lysis buffer and sonicated. The determination of protein concentration was done using the Compat-Able Protein Assay Preparation Reagent Set (Thermo Scientific, #23215, Vienna, Austria) and the Pierce BCA Protein Assay Kit (Thermo Scientific, #23227, Vienna, Austria). Samples (20 µg total protein) were separated on a 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membranes were probed with anti-perilipin antibody (Cell Signaling, #9349, Frankfurt am Main, Germany), anti-PPARγ2 antibody (Cell Signaling, #2435, Frankfurt am Main, Germany), anti-FABP4 antibody (Cayman, #10004944, Tallinn, Estonia), anti-CD24 antibody (Abcam, #179821) and anti-GAPDH (Thermo Scientific, AM4300, Vienna, Austria). Goat anti-rabbit IgG-HRP (DAKO, Vienna, Austria) and goat anti-mouse IgG-HRP (Promega, Walldorf, Germany) served as secondary antibodies and signals were detected with a chemiluminescence detection system. Membranes were stained with Ponceau S for normalization to total protein. Densitometric analysis was done using ImageJ software.
6
Virus-Like Particle ELISA Protocol
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VLP-ELISAs were performed as previously described (17 (link)). Briefly, 96well PolySorb ELISA plates (Thermo Fisher Scientific, Rockford, IL, USA) were coated with 100 ng/well purified high quality L1SHORT-VLPs in 50 mM carbonate buffer pH9.6. The next day, plates were blocked with CBB and incubated for 1 h with three-fold dilutions of Mastomys sera in CBB. Then, plates were washed four times with PBS-T and incubated with goat anti-mouse IgG-HRP (1:10,000 in CBB, Promega GmbH, Walldorf, Germany). After four washes, color development and measurement were performed as described for the GST-ELISA. Antibody titer represents the last reciprocal serum dilution above the blank.
7
Protein Expression Analysis Protocol
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To analyse protein levels, cells were harvested in SDS (sodium dodecyl sulfate) lysis buffer and sonicated. Protein concentrations were determined using the Compat-Able Protein Assay Preparation Reagent Set (Thermo Fisher Scientific, #23215, Vienna, Austria) and the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23227, Vienna, Austria) according to the manufacturer´s instructions. 20 µg of total protein per sample was separated by SDS polyacrylamide gel electrophoresis and blotted onto PVDF membranes, which were probed with anti-PPARγ (#23215, Cell Signaling), anti-FABP4 (#10004944, Cayman) or anti-DPP4 antibodies (#ab28340, Abcam). Goat anti-rabbit IgG-HRP (#W4011, Promega) and goat anti-mouse IgG-HRP (#W4021, Promega) served as secondary antibodies. Signals were detected using a chemoluminescence detection system, and membranes were stained with Ponceau S for normalization to total protein. Densitometric analysis was done using ImageJ 1.51n (NIH, USA) and Image Lab version 6.0.1 (Bio-Rad, Feldkirchen, Germany).
8
Mitochondrial Ribosomal Protein Analysis
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20–30 µg of total extracted protein was diluted to an equal volume and a ⅓ volume of NuPAGE LDS 4 x sample buffer and loaded on SDS-PAGE 4-12% bis-tris gels (Life Technologies) and transferred onto a membrane using iBlot 2 Dry Blotting System (Thermo Fisher Scientific). The following antibodies were used: rabbit anti-MRPS17 (Proteintech 18,881-1-AP, 1:1000), rabbit anti-MRPS18b (Proteintech 16,139-1-AP, 1:1000), rabbit anti-MRPS35 (Proteintech 16,457-1-AP, 1:1000), Total OXPHOS Human WB antibody cocktail (Abcam, ab110411, 1:1000), goat anti-rabbit IgG HRP (Promega W4011, 1:2000), goat, anti-mouse IgG HRP (Promega W4021).
9
SDS-PAGE and Western Blot Analysis of Shiga Toxin 2
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All gel electrophoresis equipment, buffers, gels, and PVDF membranes were purchased from Invitrogen. Proteins were separated by SDS-PAGE using 4–12% NuPAGE (denatured) Novex Bis-Tris mini gels following the manufacturer’s protocol. For Western blot analysis, proteins were electrotransferred to PVDF membranes (0.45 um). The membranes were blocked with 5% nonfat dry milk, and then probed with Stx2 mAb, Stx2-5 [26 (link)], at 20 µg/mL, or HuSAP mAb ab27313 (Abcam, Cambridge, MA, USA) at 10 µg/mL, or a mixture of mAbs Stx2-5 and ab27313, followed by goat anti-mouse IgG-HRP at 25 ng/mL (Promega, Madison, WI, USA). Bound antibody was detected using the Amersham ECL-Plus Western Blotting Detection System (GE Healthcare, UK) according to the manufacturer’s protocol.
10
Western Blot and Dot Blot Analysis
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Cell pellets (600 × g, 5 min) were lysed with RIPA buffer containing protease inhibitors (Roche). Supernatants were adjusted to 2 mg protein/mL and denatured (95°C, 5 min), and 20 μL was loaded onto the gel. After SDS-PAGE (10%, 120 V) and transfer to nitrocellulose (GE Healthcare; 300 mA, 4 hr), the membrane was blocked with 5% milk in PBST (PBS with 0.1% Tween 20), and then incubated with primary antibody overnight at 4°C. The membrane was incubated with secondary antibody for 1 hr, stained with ECL (GE Healthcare), and recorded using a digital system with Fluorochem 8900 software.
Dot blots used 1-μL samples on nitrocellulose membranes and were processed as above.
Primary antibodies were mouse monoclonals: IgG anti-β-actin (AC-74) (A2228; Sigma), IgG1 anti human p53 (2C3) (MCA35102; AbD Serotec), IgG anti-HMGB1 (H9664; Sigma), and polyclonal anti-human HSP 70 antibody (841680; R&D Systems). Dilutions were 1:1000. Secondary antibodies were as follows: goat anti-mouse IgG HRP (Promega) (1:5,000 dilution). SDS-PAGE gels were directly visualized with Instant Blue kit (Expedeon).
Dot blots used 1-μL samples on nitrocellulose membranes and were processed as above.
Primary antibodies were mouse monoclonals: IgG anti-β-actin (AC-74) (A2228; Sigma), IgG1 anti human p53 (2C3) (MCA35102; AbD Serotec), IgG anti-HMGB1 (H9664; Sigma), and polyclonal anti-human HSP 70 antibody (841680; R&D Systems). Dilutions were 1:1000. Secondary antibodies were as follows: goat anti-mouse IgG HRP (Promega) (1:5,000 dilution). SDS-PAGE gels were directly visualized with Instant Blue kit (Expedeon).
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