Antibodies against LC3B (ab48394, AbCam, Cambridge, MA, USA), p62 (ab91526), pEIF2S1 (ab32157), pJNK (sc-6254, Santa Cruz Biotechnology, Santa Cruz, CA), EIF2 (ab32157), Bax (sc493), Bcl-2 (sc492), Anti rabbit IgG (GE 30021019), Goat-anti-Mouse IgG (Zymed 626520), Rabbit-anti-Goat IgG (Zymed 811620) and Beta actin (ab8227) were used for western blotting. For immunostaining, Vectashield Mounting Medium with DAPI (Vector laboratories) and LC3B (Cell signaling 3868, Boston, MA, USA) were used. Primers for mouse GAPDH, TNFR1, IL10, IL1β, MAP1LC3, ATG5, SQSTM1, BECN-1 and ULK1 (Applied Biosystems) were used for reverse-transcriptase polymerase chain reaction (RT-PCR). Autophagy was induced with Rapamycin (Cayman Chemical Company).
Goat anti mouse igg
Manufactured by Zymo Research
Sourced in United States
Goat anti-mouse IgG is a secondary antibody used in various immunoassays, such as ELISA, Western blotting, and immunohistochemistry. It binds to the Fc region of mouse immunoglobulin G (IgG) molecules, allowing for the detection and quantification of target proteins or antigens in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab91526, by Santa Cruz Biotechnology (1 mentions)
Ab32157, by Santa Cruz Biotechnology (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Ab48394, by Abcam (1 mentions)
Sc 6254, by Santa Cruz Biotechnology (1 mentions)
Tnfr1, by Thermo Fisher Scientific (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Maxisorb plate, by Thermo Fisher Scientific (1 mentions)
Igg2c, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Image gauge v3, by Fujifilm (1 mentions)
Gapdh, by Abcom (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Complete protease inhibitors cocktail, by Roche (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Merck Group (1 mentions)
X omat film, by Kodak (1 mentions)
Anti phospho c jun ser63, by Cell Signaling Technology (1 mentions)
9 protocols using goat anti mouse igg
1
Western blot and immunostaining analysis of autophagy markers
2
Antigen-specific Antibody ELISA Protocol
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Maxisorb Plates (Nunc) were coated with 0.05 μg/well CTH522 or polyclonal goat anti-mouse IgG (Southern-Biotech), diluted 1:1000, overnight at 4 °C. Individual mouse sera were analyzed in duplicate. After blocking, serum was added in PBS with 2% BSA, starting with a 30-fold dilution for antigen-specific IgG or IgG subclasses. For analysis of total IgG2c secreted by in vitro stimulated murine B cells, the supernatant was added in 10-fold dilutions, starting from undiluted sample. HRP-conjugated secondary antibodies, goat anti-mouse IgG (Zymed), IgG2c (Invitrogen), or IgG1 (Southern Biotech), were diluted in PBS with 1% BSA. After 1 h of incubation, antigen-specific Abs were detected using TMB substrate as described by the manufacturer (Kem-En-Tec Diagnostics). ELISA data were plotted as the sum of absorbances as described previously60 (link), as a simple method to visualize antibody responses.
3
Western Blot for Protein Expression
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Whole cell extract and nuclear and cytoplasmic proteins were prepared from RAW264.7 cells by using a protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The protein concentration was measured by the Bicinchoninic acid (BCA) method. Samples (20 µg total proteins) were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon P; Millipore, MA, USA). After blocking with 5% nonfat milk in phosphate buffer solution with tween-20 (PBST) for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibody and then incubated with the conjugated secondary antibodies. The membranes were incubated with Electro-Chemi-Luminescence (ECL) detection kits for 1 min and then exposed to X-ray film. The intensity of the immunoreactive bands was determined using a densitometric analysis program (Image Gauge V3.12; Fuji Photo Film, Tokyo, Japan). Mouse monoclonal to IRF5, GAPDH and PCNA mAbs were purchased from Abcom company (Cambridge, MA, USA). Goat anti-mouse IgG was purchased from Zymed Laboratories (San Francisco, CA, USA).
4
ELISA-Based Binding Specificity and Titration of HIV mAbs
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ELISAs were performed for Ag-binding specificity analysis and titration of purified MAbs and ITs in wells coated with antigen (1 μg/ml), as described elsewhere25 . The gp41 antigen was a linear peptide HIV-1 consensus clade B sequence [LGIWGCSGKLICTT] representing the epitope of 7B2. Gp120 antigen was a recombinant protein expressed in mammalian cells. Recombinant gp120 antigen represented HIV isolate IIIB (gift from Genentech, S. San Francisco, CA). The synthetic V3 loop peptide represented the V3 sequence of strain IIIB (amino acids AA 297–330; numbering according to reference 44 (link), TRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAH. Binding of antibody to the antigen was detected with AP-conjugated secondary antibodies: goat anti-mouse IgG (H + L chain specific) for HIV MAb 924 as well as 924 based-ITs; or goat anti-human IgG (H + L chain specific) for HIV MAb 7B2 as well as 7B2 based-ITs (all from Zymed Laboratories, South San Francisco, CA). Data are reported as optical density at 405 nm and represent means of triplicate values with three independent experiments.
5
Western Blot Analysis of Chromatin Markers
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After treatment with TSA, WI-38 and HCT116 cells were harvested in lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% deoxycholate, and the cOmplete Protease Inhibitors Cocktail (Roche) and were then sonicated. Then, 30 μg of protein was loaded onto a denaturing 10-20% gradient or 16% sodium dodecyl sulfate (SDS)-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane. After incubation for 2 h in a PBS solution containing 5% albumin, the blots were exposed to the following primary antibodies: anti-HP1α (1:300); anti-HP1β (1:200); anti-H3 N-terminal (1:300); anti-H3K9me3 (1:250); anti-H3K9ac (1:250); and anti-CENP-A (1:200). The blots were incubated for 1 h at room temperature with the following horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG (1:10,000 Zymed); goat anti-rabbit IgG (1:15,000 Santa Cruz Biotechnology, Inc.); and chick anti-goat IgG (1:15,000 Chemicon International). The signal was subsequently detected by chemiluminescence (ECL kit from Millipore, USA) on Kodak X-Omat film. For the negative control, the primary antibody was omitted.
6
Immunofluorescence Staining of Immune Cells
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Mouse monoclonal antibodies included; ED1 (CD68, macrophages) and R73 (rat αβ T‐cell receptor) (Serotec, Oxford, UK), RP1 (neutrophils) (Becton Dickinson, San Diego, USA) and anti‐α‐tubulin (Abcam, Cambridge, UK). Rabbit polyclonal antibodies included anti‐phospho‐p38 Thr180/Tyr182 and anti‐phospho‐c‐Jun Ser63 (Cell Signaling, Boston, MA, USA) and goat anti‐γ‐fibrinogen (Santa Cruz biotechnology, CA, USA). Biotinylated antibodies included goat anti‐mouse IgG and goat anti‐rabbit IgG (Zymed, San Francisco, CA, USA). Immunofluorescence staining used FITC (Fluorescein isothiocyanate)‐conjugated rabbit polyclonal antibodies against sheep IgG Dako, Glostrup, Denmark, rat IgG (Sigma‐Aldrich, Castle Hill, NSW, Australia) and rat C3 (Cappel, Malvern, PA, USA).
7
Antibody Panel for Rat Immune Profiling
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Mouse antirat monoclonal antibodies used in this study included ED1 (CD68), RECA-1 (endothelium), CD161 (NK cells), R73 (rat αβ T-cell receptor) (Serotec, Oxford, UK), as well as RP1 (neutrophils) (Becton Dickinson, San Diego, CA, USA). Other antibodies included rabbit antifibrinogen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fluorescein isothiocyanate-conjugated goat polyclonal antibodies against rat immunoglobulin G; (IgG; Sigma Aldrich, St. Louis, MO, USA), rabbit anti-C4d (Hycult Biotech, Plymouth Meeting, PA, USA), and Alexa594 conjugated donkey antirabbit IgG (Invitrogen, Carlsbad, CA, USA). Biotinylated secondary antibodies were goat antimouse IgG (Zymed, San Francisco, CA, USA) and goat antirabbit IgG (Invitrogen), which were detected using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Other secondary antibodies included horseradish peroxidase-conjugated goat antirabbit IgG, goat antimouse IgG, and mouse peroxidase-conjugated antiperoxidase complexes (all from Dako, Glostrup, Denmark). Tacrolimus was obtained in powder form from Selleck Chemicals (Houston, TX, USA).
8
Autophagy Pathway Regulation Analysis
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AA and Compound C (C.C) were purchased from Calbiochem (San Diego, CA, USA). Anti-phospho-ACC, phospho-LKB1, procaspase-3, PARP, BclXL, LC3 I/II, beclin-1, AMPK, and phospho-AMPK antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Bal-A1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, rabbit anti-goat, and goat anti-mouse IgGs were obtained from Zymed Laboratories (San Francisco, CA, USA). FX, acrydine orange hemi zinc chloride salt, 3-methyladenine (3-MA), anti-ß-actin antibody and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Anticancer compound analysis protocol
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Anti-PARP, anti-caspase 3, anti-ACC, anti-p-AMPK, anti-AMPK, anti-p-mTOR, anti-mTOR, anti-p-P70S6K1, anti-P70S6K1, anti-cyclin D1, anti-cyclin E1, anti-p53 and ant-β actin antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-CDK2, anti-CDK6, anti-Bcl2, anti-BAX, and p21 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Zymed Laboratories (San Francisco, CA, USA). The compound C was purchased from Calbiochem (San Diego, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI), JC-1 and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HsA was kindly provided by Prof. Jong Rok, Lee (Daegu Haany University, Gyeongsan, Korea), and isolated as previously described [23 (link)]. The pure HsA (1.31 g) was isolated from the chloroform extract (150 g) of H. lyrata, and was stored at −20 °C. The purity (>97%) was verified by comparing retention time with standard compounds.
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