Immunoprecipitation (IP) of IgG was performed according to manufacturer's protocol (Bio‐Rad). In brief, protein G‐coated magnetic beads (SureBeads™ Protein G Magnetic Beads; Bio‐Rad, 161‐4023) were washed with PBS‐T (PBS pH 7.4 and 0.1% Tween 20; EMD Millipore, 9005‐64‐5) and incubated with 1 μg of goat anti‐human IgG antibody (EMD Millipore, AP112, 1:400) for 1 hour. IgG‐coupled beads were incubated o/n with 15 μg protein from tissue lysates diluted in PBS. Magnetic beads were washed with PBS and dissolved in 40 μL Laemmli Buffer and 1% Nu‐Page sample reducing agent (Invitrogen, NP0004) and incubated for 10 minutes at 70°C. The precipitate was collected and used for gel electrophoresis and Western blotting (WB). Total of 15 μg protein per sample was loaded on pre‐casted Bolt 4%‐12% Tris‐Plus Gels (Invitrogen, NW04120BOX) for 1 hour at 160 V in MOPS SDS running buffer (Invitrogen, NP0001‐02). Proteins were transferred to PVDF membranes (Millipore, IPVH00010) and incubated o/n with a primary antibody (mouse anti‐human IgG; Novus, IG226, 1:400) and 1 hour with a secondary HRPO polyclonal Rabbit antimouse IgG (Dako, P0260, 1:2000). For visualization, a chemiluminescent peroxidase substrate (Sigma, CPS1120) was used and images were quantified using Image Lab Software (Bio‐Rad, 5.1 V).
Rabbit anti mouse igg
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Rabbit anti-mouse IgG is a secondary antibody used in various immunoassays and immunochemical techniques. It is specifically designed to recognize and bind to mouse immunoglobulin G (IgG) antibodies, allowing for the detection and quantification of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Agilent Technologies (4 mentions)
Avidin biotin complex abc, by Agilent Technologies (2 mentions)
3 3 diaminobenzidine (dab), by Merck Group (2 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Surebeads protein g magnetic beads, by Bio-Rad (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Polystyrene microtiter plate, by Corning (1 mentions)
Phospho h2ax ser139, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Agilent Technologies (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Gamma counter, by PerkinElmer (1 mentions)
Triton x 100, by Carlo Erba (1 mentions)
α btx, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Deadend colorimetric tunel system, by Promega (1 mentions)
Sepharose beads, by Merck Group (1 mentions)
Ucd 300, by Diagenode (1 mentions)
Gammabind g sepharose beads, by GE Healthcare (1 mentions)
Anti ets2 sc 351, by Santa Cruz Biotechnology (1 mentions)
16 protocols using rabbit anti mouse igg
1
IgG Immunoprecipitation and Western Blotting
2
Serodiagnosis of Sparganosis Using ELISA
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Polystyrene microtiter plates (Costar, Corning, New York, USA) were coated with 0.5 µg/well (100 µl/well) of rSepCp-1 protein or sparganum crude extract (SeC) diluted in 0.1 M carbonate/bicarbonate buffer, pH 9.6, and incubated at 4℃ overnight. After washing 3 times with PBST, plates were blocked with 1% bovine serum albumin for 1 hr at 37℃. After washing, 100 µl of human or mouse serum samples diluted 1:500 were loaded into each well of the plates, and the plates were incubated for 2 hr at 37℃. Then, 100 µl of horseradish peroxidase-conjugated goat anti-human IgG (1:10,000) (ICN Pharmaceuticals Inc., Aurora, Ohio, USA) or rabbit anti-mouse IgG (1:5,000) (DakoCytomation) was loaded into each well. After incubation for 1 hr at 37℃, the reaction was developed with 1-Step™ Ultra TMB-ELISA substrate (Pierce) at room temperature, and the absorbances were read at 450 nm. All samples were run in duplicate. The cut-off values were defined as the mean absorbance plus 3 SDs of the values obtained from the sera of healthy negative controls.
3
Antibody Detection of Apoptosis Markers
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Antibodies against cleaved caspase 3, phospho-p53 (Ser15), p53, cleaved PARP (Asp214), β-actin, phospho-H2AX (Ser139) were obtained from Cell Signaling (Beverly, MA, USA). NOXA was purchased from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA, USA). As secondary antibodies horseradish peroxidase-conjugated goat-anti-rabbit (Sigma), horseradish peroxidase-conjugated rabbit anti-goat or rabbit anti-mouse IgG from Dako (Glostrup, Denmark) were used.
4
Quantifying Anti-Acetylcholine Receptor Antibodies
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Acetylcholine receptor-specific antibodies were determined in serum from individual mice by a radioimmunoprecipitation assay (67 (link)). Mouse AChR was extracted from whole carcass, as previously described, and labeled with 2 nM [125I]α-BTX. Serum samples were incubated overnight with [125I]-αBTX labeled mouse AChR (0.5 pM). Antibody–AChR complexes were precipitated by adding an excess of rabbit anti-mouse IgG (DAKO). Pellets were washed twice with cold PBS plus 0.5% Triton X-100 (Carlo Erba) and [125I]-αBTX labeling was assessed using a γ-counter (PerkinElmer). Serum samples incubated with mouse AChR and pre-incubated in excess of unlabeled α-BTX (Life Technologies) (unspecific binding) were subtracted from test samples. The anti-AChR antibody titers were expressed as pM of [125I]α-BTX-binding sites precipitated per milliliter of serum.
5
Silibinin Modulates Tumor Markers
6
ChIP-qPCR Assay for ETS2 Transcription Factor
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HeLa cells were fixed in medium with 1% formaldehyde for 20 min and then harvested in SDS lysis buffer (50 mM Tris HCl pH 8.1, 10 mM EDTA, and 1% SDS) with added protease inhibitors. Chromatin was then sonicated using 25 cycles of 30 s ON/OFF on a Diagenode UCD-300 sonicator. Sheared chromatin was pre-cleared using sepharose beads (Sigma Aldrich) followed by overnight incubation with 5 μg anti-ETS2 (sc-351, Santa Cruz Biotechnology) or 5 μg of rabbit anti-mouse IgG (Dako) as a background control. Samples were then incubated with blocked GammaBind G sepharose beads (GE Healthcare Life Sciences, Freiburg, Germany) and eluted in elution buffer (0.1M NaHCO3, 1% SDS). Untreated chromatin was used as input. Purified DNA was used in qPCR reactions for analysis. All primers are listed in Table S4 in Supplementary Material.
7
Immunohistochemical Staining Protocol
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Sections were deparaffinized, hydrated, and boiled in 10 mM citrate buffer (pH 6) for 20 min for antigen retrieval, rinsed in Tris-buffered saline (TBS, pH 7.6, 0.05 M), and incubated in the primary antibodies overnight in a humid chamber. After rinsing, they were incubated in the corresponding biotinylated secondary antibodies (rabbit anti-mouse IgG or goat anti-rabbit IgG; Dako, Glostrup, Denmark), diluted at 1:200 in TBS, followed by incubation with avidin-biotin complex (ABC, DAKO) in TBS. Bound peroxidase was revealed using 0.04% 3,3-diaminobenzidine (Sigma, USA), 0.05% ammonium nickel (II) sulfate, and 0.03% hydrogen peroxide in TBS, pH 7.6. Sections were dehydrated, cleared, and coverslipped using Eukitt (O. Kindler, Freiburg, Germany). Negative controls omitted the primary antibodies.
8
Immunohistochemical Detection of Tau Pathology
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Tissue sections were treated as described above. An additional blocking step before the addition of milk was used to reduce endogenous peroxidase activity, using methanol containing 0.3% hydrogen peroxide. Sections were incubated with the respective primary antibody, AT8 (Thermoscientific, 1:600), AT100 (Thermoscientific, 1:500), AT180 (Thermoscientific, 1:500), PHF1 (source: Peter Davies; 1:500) or Aβ-XP (Cell Signaling, 1:200), prior to tracer labelling. Incubation with a secondary biotinylated antibody (either rabbit anti-mouse IgG or swine anti-rabbit IgG; Dako, 1:200, 30 min) was followed by treatment with avidin-biotin complex (Vector Laboratories; 30 min). Tyramine signal amplification (red or green; 1:200) was used as a substrate for horseradish peroxidase. Sections were mounted with either DAPI-containing fluorescent mounting medium or Nissl Neurotrace 640, as described above.
9
ADAM10 Expression in Colorectal Cancer
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Formalin-fixed, paraffin-embedded sections from available Crc specimens and normal mucosa underwent antigen retrieval and quenching with 3% hydrogen peroxide. Sections were incubated 12 hr at 4°C with anti-ADAM10 N-terminal antibodies (1:500 Sigma; 1:1000 ab39153, Abcam), followed by labelled Envision anti-rabbit System (Dako). The immunoreaction was revealed by HRP using 3,3′diaminobenzidine (Biogenex), the slides were slightly counterstained with Harris's hematoxylin, analysed with optical microscope Axioskope 2 (Zeiss) and images acquired with AxioVision 4.4 system (Zeiss). Available tumor specimens frozen at −80°C were mechanically broken and incubated with 1%TritonX-extraction buffer containing protease- and phosphatase-inhibitors (Sigma). Proteins were resolved by SDS-PAGE and WB analysis was performed with anti-ADAM10 (AB936, R&D-Systems) and anti-β-actin (Sigma) Abs. Secondary Abs were HRP-conjugated goat-anti-rabbit IgG and rabbit-anti-mouse IgG (Dako). The protein loading was normalized by ponceau-staining and β-actin immunostaining.
10
Quantification of Plasma Protein Interactions
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Microtitier plate Maxisorp (Nunc) was coated with H. alvei PCM 1200 LPS (5 μg/well), and then after blocking with 1% BSA in TBS/Ca2+, 500 ng of plasma-derived ficolin-3 complexes in MBL-binding buffer (20mM Tris, 1M NaCl, 10mM CaCl2, pH 7,4) supplemented with 0,1% BSA was added and incubated overnight at 4°C. After washing in TBS/Ca2+, antibodies IgA, IgG and IgM were detected by HRP-conjugated primary antibodies (as above); and ficolin-3, thrombin, C1q and CL-11 were detected by following primary Ig: (i) anti-ficolin-3 mouse monoclonal IgG1 (clone 4H5; Hycult Biotech), (ii) anti- thrombin rabbit polyclonal (catalogue number: ab92621, Abcam, UK), (iii) anti-Complement 1q (C1q) goat polyclonal (catalogue number: 234390, Calbiochem, USA), (iv) anti-collectin-11 (CL-11) rabbit polyclonal (catalogue number: HPA035241, Sigma-Aldrich) and HRP-conjugated secondary antibodies: rabbit anti-goat IgG (catalogue number: P0448, Dako) or goat anti-rabbit IgG antibodies (catalogue number: P0448, Dako) or rabbit anti-mouse IgG (catalogue number: P0260, Dako).
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