Zygotes were lysed with SDS sample buffer (100 zygotes per sample) and heated for 5 min at 95 °C. Total zygotes proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA), followed by blocking in TBST containing 5% defatted milk (BD, USA) for 30 min. First, the membranes were incubated with primary antibodies overnight at 4 °C. Then, the membranes were washed in TBST, and incubated with a HRP-linked secondary antibody for 1 h at room temperature, followed by washing with TBST three times. Finally, bounding antibodies were detected using SuperSignal WestFemto maximum sensitivity substrate (Thermo Fisher, USA). The primary antibodies used and dilution factors are anti-DDB1 (Epitomics, #3821-1, 1:10000) and anti-FLAG antibodies (Sigma, #F3165, 1:3000). The secondary antibody was HRP-conjugated anti-rabbit/mouse IgG (Jackson ImmunoResearch Laboratories).
Hrp conjugated rabbit anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
HRP-conjugated rabbit anti-mouse IgG is a secondary antibody used for detection in various immunoassays. It is produced by immunizing rabbits with mouse immunoglobulin G (IgG) and conjugating the resulting antibodies with horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
Exoab cd63a 1, by System Biosciences (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Anti ddb1, by Abcam (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Defatted milk, by BD (1 mentions)
Cleaved il 1β, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Bca assay, by Boster Bio (1 mentions)
Tanon 5200 chemiluminescent imaging system, by Shanghai Tanon Science & Technology (1 mentions)
Fitc conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Hrp conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Enhanced chemiluminescence ecl reagent, by GE Healthcare (1 mentions)
Streptavidin fitc, by Merck Group (1 mentions)
Pflag ctc vector, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
β mercaptoethanol, by Bio-Rad (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
8 protocols using hrp conjugated rabbit anti mouse igg
1
Western Blot Analysis of Zygotic Proteins
2
Western Blot Analysis of Cellular Signaling
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The cultured cells or skin tissues were lysed in RIPA lysis buffer with protease and phosphatase inhibitors. The protein concentration was quantified by bicinchoninic acid (BCA) assay (Boster, California, United States). The protein samples were resolved by 10% SDS-PAGE gels and then transferred to PVDF membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill, County Cork, Ireland). After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight. The membranes were then incubated with HRP-conjugated anti-rabbit/mouse IgG (111-035-003/115-035-003, Jackson ImmunoResearch, West Grove, United States) secondary antibodies for 1 h at room temperature. Images were visualized on Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology).
3
Investigating Cell Adhesion Molecules
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MOPC-21 (IgG1), pFLAG-CTC vector, and Streptavidin-FITC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD44 (clone 156-3C11) and anti-α2 integrin subunit (clone P1E6, IgG1) antibodies were purchased from Millipore (Billerica, MA, USA). FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rabbit IgG, HRP-conjugated rabbit anti-mouse IgG, HRP-conjugated rat anti-rabbit IgG, and HRP-conjugated Streptavidin were purchased from Jackson Immunoresearch (West Glove, PA, USA). Anti-CD44v10 antibody (AB2082) was purchased from Calbiochem (San Diego, CA, USA). Anti-FLAG (M2) antibody was purchased from Stratagene (Santa Clara, CA, USA). Type I Collagen was purchased from Inamed Biomatenak (Freemont, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from GE Healthcare (Piscataway, NJ, USA). Other chemicals were purchased from Sigma-Aldrich unless otherwise mentioned.
4
Western Blotting of Exosomal CD63
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For Western blotting, 4 μL of Type A exosomes were mixed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), then with Laemmli sample buffer containing β-mercaptoethanol (Bio-Rad, Oxford, UK), denatured at 95 °C for 5 min, loaded to Mini-PROTEANR TGX™ 10% gradient SDS-PAGE gels (Bio-Rad, Oxford, UK), and blotted on Immobilon-P membranes (Millipore, Bedford, MA, USA). Blocking (1 h, RT) and antibody incubations (the primary antibodies overnight at 4 °C; the secondary antibodies 1.5 h at RT) were performed in 5% non-fat powdered milk (Valio, Helsinki, Finland) in TBS and in TBS with 0.1% Tween-20, respectively. The anti-human CD63 antibody (EXOAB-CD63A-1; 1:1000) was purchased from System Biosciences (Palo Alto, USA), and the anti-bovine CD63 antibody (abx021473; 1:500) from Abbexa (Cambridge, UK). For detection, horseradish (HRP)-conjugated secondary antibodies (HRP-conjugated rabbit anti-mouse IgG, 1:20,000; HRP-conjugated goat anti-human IgG (for detection of T-DM1), 1:20,000, both from Jackson ImmunoResearch; and HRP-conjugated goat anti-rabbit IgG, 1:20,000, System Biosciences) and a 1:2 mixture of SuperSignal™ West Pico Chemiluminescent Substrate and SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA) were used.
5
Screening and Characterization of scFv Antibodies
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The EV71-infected lysates were separated on 15% SDS-PAGE under reducing conditions, which were transferred into PVDF membranes. After blocking with 5% skimmed milk in PBS at room temperature (25 °C) for 1 h, the scFv antibodies were detected using mouse anti-HA tag (Proteintech, Rosemont, IL, USA) and HRP-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) antibodies. The membranes were developed using the 3,3ʹ-diaminobenzidine (DAB) reagent. Similarly, the IgY was detected using HRP-conjugated donkey anti-chicken IgY (Jackson ImmunoResearch, West Grove, PA, USA). For the cross-reactivity assay, the selected scFv antibodies (10 μg/mL) were added and detected by mouse anti-HA tag and HRP-conjugated rabbit anti-mouse. Other steps, such as blocking, washing, incubation and color development steps were performed as described above.
6
Western Blot Analysis of Nectin-4
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Purified proteins or whole cell lysates were analyzed using standard western blot analysis. Briefly, samples were separated by SDS-PAGE, and the gel was then stained with Coomassie blue for protein visualization or transferred to a polyvinylidene fluoride (PVDF) membrane for blocking and antibody incubations. For the detection of recombinant nectin-4, the membrane was incubated with mouse anti-His IgG (1:3000; Bioman Scientific, New Taipei City, Taiwan) and secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:5000; Jackson ImmunoResearch, West Grove, PA, USA). The membrane was then visualization by 3, 3’-diaminobenzidine tetrahydrochloride (DAB) staining. For the detection of endogenous nectin-4 from whole cell lysates using scFv, the membrane was incubated with scFv (10 µg/ml), mouse anti-HA secondary antibody (1:5000; Cat# 66006-1, Proteintech, Rosemont, IL, USA), and HRP-conjugated anti-mouse tertiary antibody (1:5000; Cat# 7076, Cell Signaling Technology, Danvers, Massachusetts, USA). Finally, the membrane was stained with Clarity Western ECL Substrate (Bio-Rad) and visualized by ImageQuant™ LAS 4000 (GE Healthcare Life Science).
7
Quantifying Endothelial Cell Activation
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Confluent monolayers of HUVECs in 96-well microplates were incubated with NETs for 6 hours. Some cultures were additionally supplemented with defibrotide. Cells were fixed by adding an equal volume of 8% paraformaldehyde for 30 minutes. Blocking was with 2× blocking solution (ab111541, Abcam) at room temperature for 2 hours. After washing with PBS, cells were incubated with 5 μg/mL primary mouse anti-human antibodies against E-selectin (BBA26, R&D), VCAM-1 (BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight. Next, 100 μL of diluted HRP conjugated rabbit anti–mouse IgG (1:2000, Jackson ImmunoResearch, catalog 315-035-003) in 1× blocking solution was added and incubated at room temperature for 1 hour. After washing thoroughly with PBS, 100 μL of TMB substrate was added, and blue color development was measured at OD 650 nm with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). The signals were corrected by subtracting the mean signal of wells incubated in the absence of the primary antibody.
8
Functional Analysis of LMF1 Variants
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