Mouse anti-GFP (mix of clones 7.1 and 13.1, Roche) (1:1000), rabbit anti-Vps35 (GTX108058, GeneTex) (1:1000), mouse anti-EGFR antibody (BD Transduction Laboratories) (1:200), HRP-labeled secondary antibodies (Sigma-Aldrich) (1:5000), rabbit-anti EEA1 antibody (Enzo) (1:100), fluorescently labeled secondary antibodies (Jackson ImmunoResearch Laboratories) (1:200), Hoechst (1:2500), Human EGF (Sigma). Mouse antibodies against SNX3 (C-16 Santa Cruz Biotechnology) (1:200).
Hrp labeled secondary antibody
Manufactured by Merck Group
Sourced in United States
HRP-labeled secondary antibodies are laboratory reagents used in various immunoassay techniques. They consist of polyclonal or monoclonal antibodies that are conjugated with the enzyme horseradish peroxidase (HRP). These antibodies can bind to primary antibodies and facilitate the detection of target analytes through colorimetric or chemiluminescent reactions.
Automatically generated - may contain errors
Lab products found in correlation
Image lab, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Tmb 3 39 5 59 tetramethylbenzidine, by BD (2 mentions)
Human egf, by Merck Group (1 mentions)
Fluorescently labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Hoechst, by Merck Group (1 mentions)
Rabbit anti eea1 antibody, by Enzo Life Sciences (1 mentions)
Mouse monoclonal anti rhoa antibody, by Cytoskeleton (1 mentions)
Mem per eukaryotic membrane protein extraction reagent kit, by Thermo Fisher Scientific (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Gst rbd beads, by Cytoskeleton (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Streptavidin agarose beads, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Smad4, by Abcam (1 mentions)
P smad2 3, by Abcam (1 mentions)
Immobilon western hrp substrate kit, by Merck Group (1 mentions)
Infinite f500, by Tecan (1 mentions)
Ab109125, by Abcam (1 mentions)
12 protocols using hrp labeled secondary antibody
1
Immunofluorescence Staining of Cells
2
GPR56 Receptor Characterization and Protein Interactions
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Sulfo-NHS-Biotin reagent was purchased from Pierce; Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit was from Thermo Scientific; Streptavidin–agarose beads from Sigma; RIPA buffer from Boston Bioproducts; Protease inhibitor cocktail (EDTA-free) from Roche Diagnostics; Collagen III protein was from AbCam; Mouse GPR56 cDNA cloned into pCDNA3.1(+) vector as described previously [11] (link); GPR56 mutations were created by site-directed mutagenesis using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene), as previously described [12] (link); Cholera toxin B subunit (CTB)–Alexa Fluor 488 were purchased from Invitrogen. Rabbit polyclonal anti-CTB antibody was generated in the Lencer lab [13] (link). HRP-labeled secondary antibodies were purchased from Sigma-Aldrich. Alexa Fluor 488 goat anti-mouse IgG (H+L) was purchase from Invitrogen. Mouse anti-GPR56N (CG4) was purchased from Biolegend. The mouse anti-GPR56N (H11) antibody was generated at the Dana Farber/Harvard Cancer Center Monoclonal Antibody Core and the rabbit anti-GPR56C (199) antibody was generated at Yenzym Antibodies, as previously described [14] (link), [15] (link). The GST-RBD beads and mouse monoclonal anti-RhoA antibody were purchased from Cytoskeleton.
3
Western Blot Analysis of NME2 Protein
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H1299 and H157 cells were collected at indicated times. Then, extracted proteins were separated using SDS-PAGE. Separated proteins were transferred onto PVDF membrane (Millipore, USA), which were then cultured with 5% non-fat milk in TBST for 1 h. The protein of interest was incubated with NME2 primary antibody (Abcam, USA) at 4°C for 12 h, and continued to be incubated with secondary antibodies (HRP-labeled secondary antibodies, Sigma, USA) for 2 h at 37°C. The reaction was visualized using ECL (Millipore, USA).
4
Quantitative Protein Expression Analysis
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Cells were routinely harvested as indicated time, and the irradiated cells were harvested at 24 h after radiation. Protein lysates were separated in SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in TBS buffer for 1 h, and then incubated with primary antibodies overnight at 4 °C. At room temperature, the membranes were incubated with HRP-labeled secondary antibodies (Sigma, USA) for 2 h. The reaction was visualized using Immobilon™ Western HRP substrate kit (Millipore, USA). The primary antibodies were as follows: TGF-β2 and c-Myc (Santa Cruz, USA), TGF-βRIII (CST, USA), ARHGEF15 (Abcam, USA), ABL2 (Abcam, USA), p-SMAD2/3 (Abcam, USA), E2F6 (Abcam, USA), SMAD4 (Abcam, USA), GAPDH (CST, USA). SMAD4 protein expression in pancreatic cells was tested by western blot (Additional file 4 : Figure S3B).
5
Protein Expression Analysis in Cells
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Total proteins harvested from cells and tumor samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (Polyvinylidene Fluoride) membranes. Then, the membranes were blocked with 5% skimmed milk and incubated with the following specific primary antibodies at 4 °C overnight: anti-HOXB4, anti-E-cadherin, anti-Vimentin, anti-Snai1, and anti-Zeb1 antibody. GAPDH was used as a loading control. After washing with PBST, the membranes were incubated with HRP-labeled secondary antibodies (Sigma, USA). Protein intensity was detected by Image Lab (Bio-Rad, USA).
6
Western Blot Analysis of EMT Markers
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Total proteins harvested from cells and tumor samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Then, the membranes were blocked with 5% skimmed milk and incubated with the following speci c primary antibodies at 4 °C overnight: anti-HOXB4, anti-E-cadherin, anti-Vimentin, anti-Snai1, and anti-Zeb1 antibody. GAPDH was used as a loading control. After washing with PBST, the membranes were incubated with HRP-labeled secondary antibodies (Sigma, USA). Protein intensity was detected by Image Lab (Bio-Rad, USA).
7
Protein Expression Analysis in Cells
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Total proteins harvested from cells and tumor samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (Polyvinylidene Fluoride) membranes. Then, the membranes were blocked with 5% skimmed milk and incubated with the following speci c primary antibodies at 4°C overnight: anti-HOXB4, anti-E-cadherin, anti-Vimentin, anti-Snai1, and anti-Zeb1 antibody. GAPDH was used as a loading control. After washing with PBST, the membranes were incubated with HRP-labeled secondary antibodies (Sigma, USA). Protein intensity was detected by Image Lab (Bio-Rad, USA).
8
Quantification of Cell Surface Receptors
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Cell surface receptor quantification was performed as previously described [17 (link)]. Briefly, HEK293T/17 cells were split into 24-well plates pre-coated with Poly-D-lysine, transiently transfected with a control empty vector (pcDNA3.1) or increasing amounts of vector encoding N-terminally Myc-tagged P2Y1-R or HA-tagged AT1-R. Forty-eight hours post-transfection, cells were fixed (1% paraformaldehyde), saturated (PBS–1% BSA) and incubated with the primary anti-Myc antibody (Clone 9E10. 1:500. Santa Cruz Biotechnology. Dallas, Texas, USA) or anti-HA (Clone 16B12. 1:2500. BioLegend. San Diego. California, USA) and then with HRP-labeled secondary antibody (Sigma-Aldrich/Merck. 1:1000. Darmstadt, Germany). After washing, cells were incubated for 15 min with HRP substrate: TMB (3,39,5,59-tetramethylbenzidine) (BD Biosciences). The reaction was stopped with HCl 1N, and the plates were read at 450 nm in a microplate reader (Infinite F500. Tecan Group Ltd. Männedorf, Switzerland).
9
Transient Transfection and Immunodetection
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WTT-CHO cells were transiently transfected in 100-mm Petri dishes with pcDNA3.1 (+) empty vector (control) or vectors encoding N-terminally HA-tagged receptors. Eighteen hours post-transfection, 120,000 cells/well were then split into 24-well plates precoated with poly-D-lysine. Cells were then fixed (4% paraformaldehyde), saturated (PBS - 1% BSA) and incubated with the primary anti-HA antibody (BioLegend, 16B12 Clone) and then with the HRP-labeled secondary antibody (Sigma, St. Louis, MO, USA). After washing, the cells were incubated for 1 h with the TMB (3,39,5,59-tetramethylbenzidine) HRP substrate. The reaction was stopped with 1 N HCl, and the plates were read at 450 nm in a microplate reader (Tecan Infinite F500).
10
Protein Expression Analysis via Western Blot
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Western blot (WB) assays was performed as previously described (30 (link)). Briefly, we prepared cell extracts for Western blotting in RIPA buffer. Then, lysates were separated by SDS-PAGE and were transferred to PVDF membranes (Millipore, Billerica, MA). Primary antibodies PDPN (Abcam, ab236529,1:1000), TIMP1 (Abcam, ab109125,1:1000), EMP3 (Santa cruz, sc-81797, 1:100), TAGLN2 (Proteintech, 10234-2-AP, 1:200), and GAPDH (Abcam, ab181602, 1:10000) were used along with HRP-labeled secondary antibody (1:10000, Sigma) in Western blot. The immune complex was detected by chemiluminescence (GE Healthcare, Wauwatosa, WI).
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