Sixty microgram of protein from the pooled serum of 8 patients with MM and eight healthy controls was separated in NuPAGE Novex 4–12% bis-tris gradient gels (Invitrogen) and transferred to a nitrocellulose membranes (Millipore, Billerica, MA, USA) for 30 min using 1 × NuPAGE transfer buffer. nitrocellulose membranes were blocked overnight at 4°C in blocking buffer (5% skim milk, 0.1% Tween 20, 0.01 mol/L PBS). After washes with washing buffer (0.1% Tween, 0.01 mol/L PBS), the membranes were incubated with primary antibodies in the blocking buffer for 120 min. Antiantibodies antibodies made against specific to integrin alpha-1 and isoform-1 of multimerin-1 (Abcam, Cambridge, MA, USA) were used at 1:1000 and 1:2000 dilution, respectively. After washing, the membrane was incubated with second antibodies (goat anti-mouse/HRP Conjugate, Santa Cruz, Paso Robles, CA, USA) for integrin alpha-1 and isoform-1 of multimerin-1 and detected with the Pierce chemiluminescent (ECL) kit (Thermo, Waltham, MA, USA). Levels of proteins were then quantified by Image Quant TL Activation Wizard software, USA.
Goat anti mouse hrp conjugate
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-mouse-HRP conjugate is a secondary antibody that binds to mouse primary antibodies. The horseradish peroxidase (HRP) enzyme is conjugated to the goat anti-mouse antibody, allowing for colorimetric or chemiluminescent detection of target proteins in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Nupage novex 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp conjugate, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic mixture, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Trypan blue vital stain, by Thermo Fisher Scientific (1 mentions)
Celastrol, by Alexis Biochemicals (1 mentions)
6 protocols using goat anti mouse hrp conjugate
1
Quantifying Serum Protein Biomarkers in MM
2
Immunoassay Antibody Validation Protocol
3
Celastrol's Anti-cancer Potential Revealed
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Celastrol with purity greater than 98% was purchased from Alexis Biochemicals (San Diego, CA, United States). RPMI 1640, 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture were obtained from Invitrogen (Carlsbad, CA, United States). Propidium iodide (PI), MTT, Tris, glycine, NaCl, SDS, BSA, and β-actin antibody (mouse monoclonal) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States). FBS was purchased from BioWest (Miami, FL, United States). Bortezomib (Velcade, PS341) was purchased from LC Laboratories (Woburn, MA, United States). Nuclear extraction and DNA binding kits was obtained from Active Motif (Carlsbad, CA, United States). Caspase-Glo 3/7 Assay kit was purchased from Promega (Madison, WI, United States). Rabbit polyclonal antibodies against Poly(ADP-ribose) polymerase (PARP), MMP-9, caspase-3, and goat anti-rabbit-horse radish peroxidase (HRP) conjugate and goat anti-mouse HRP conjugate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Rabbit polyclonal antibody against CXCR4 was purchased from Abcam (Cambridge, MA, United States). CXCL12 was purchased from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). ELISA kits for mouse IL-6 and TNF-α were purchased from R&D Systems Inc. (Minneapolis, MN, United States).
4
Exosome Protein Analysis by Western Blotting
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Lysates of sucrose cushion-purified Du145 exosomes or whole cells, made using RIPA buffer (Santa Cruz), were boiled in SDS sample buffer containing 20 mm DTT as previously described (29 (link)) and prepared as matched protein doses (12.5 μg to 50 μg per lane). They were subjected to electrophoresis on NuPAGE precast 4–20% gradient gels (Invitrogen). Samples were transferred to PVDF membranes before blocking in PBS containing 0.5% (w/v) Tween-20 and 3% (w/v) nonfat powdered milk. Membranes were probed with antibodies including DAF (CD55) (Diaclone, Besancon Cedex, France), Notch 3, ADAM9 (R&D Systems), RAC1 (Becton Dickinson), Tissue Factor, TSG101, calnexin (Santa Cruz), or L1CAM (a gift from P. Altevolgt, Heidelberg, Germany). After incubation with goat anti-mouse-HRP conjugate (Santa Cruz), bands were detected using x-ray film (GE Healthcare) and a chemiluminescence reagent (Super Signal West Pico, Thermo/Pierce).
5
Protein Extraction and Western Blot Analysis
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Whole protein lysates from both 2-D and 3-D cultures were extracted using RIPA buffer under standard conditions. Protein concentrations were determined using the detergent-compatible, BCA Protein Assay Kit (Pierce, Grand Island, NY; 23227). Protein lysates were separated using SDS-PAGE electrophoresis and transferred to nitrocellulose membranes for probing. The monoclonal mouse antibody, DO-1 (SantaCruz; sc-126, dilution 1:200) was used to probe full-length p53 protein, while M168 antibody (Abcam, Cambridge, ab76055, dilution 1:200) was used to probe E-cadherin. The blots were then washed and incubated with a goat anti-mouse-HRP conjugate (SantaCruz; sc-2005, dilution 1:1000). After probing, blots were treated with either Amersham ECL Detection Reagent (GE Healthcare, RPN2209) or SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher; 34095) and imaged using a Bio-Rad ChemiDoc™. Blots were then stripped and re-probed with GAPDH primary antibody (SantaCruz; sc-32233) as a positive loading control.
6
THP-1 Cells Infection and Western Blot
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