Cortical tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma) supplemented with protease (Complete Mini, Roche, Basel, Switzerland) and phosphatase (PhosStop, Roche) inhibitors according to standard protocols. Western blot analyses were conducted according to standard protocols. Briefly, soluble extracts were loaded onto Criterion, 4%–15% Tris-HCI 4 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad, Berkeley, CA, USA), separated at 120 V, and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) at 30 V for 2 h or overnight at 4 °C. Membranes were blocked with 5% BSA/1X TBS-T (Tris-buffered saline with 0.1% Tween 20) for 1 h at room temperature, and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, and secondary antibodies (1:5000 dilution; IR-Dye antibodies, LI-COR, Lincoln, NE, USA) for 1 h at RT. Membranes were washed in 1X TBS-T and scanned using the Odyssey Infrared Imaging System (LI-COR). Primary antibodies were used as follows: goat anti-Gli3 (1:500; R&D Systems) and α-Tubulin (1:5000, Abcam). Quantification and analysis were conducted using the Odyssey System and Image Studio Software version 4.0.21 (LI-COR, Lincoln, NE, USA).
Irdye antibodies
Manufactured by LI COR
Sourced in United States
IRDye antibodies are fluorescent-labeled antibodies designed for use in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry. These antibodies are available in a range of near-infrared (NIR) dye options, providing researchers with flexible labeling solutions for their specific experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (6 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Goat anti gli3, by R&D Systems (2 mentions)
α tubulin, by Abcam (2 mentions)
Image studio software version 4, by LI COR (2 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (2 mentions)
Odyssey system, by LI COR (2 mentions)
Phosstop, by Roche (2 mentions)
Complete mini, by Roche (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (2 mentions)
Yn 20, by Santa Cruz Biotechnology (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Rolipram, by Bio-Techne (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Mouse anti gapdh antibody, by Merck Group (1 mentions)
Dyngo 4a, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
F1804, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
21 protocols using irdye antibodies
1
Western Blot Protocol for Protein Analysis
2
Signaling Pathway Characterization Protocol
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(-)-Isoproterenol hydrochloride was purchased from Sigma-Aldrich (Cat #I6504), dissolved in 100 mM ascorbic acid to 10 mM stock, and used at 1 μM final concentration. Arginine vasopressin acetate salt was purchased from Sigma-Aldrich (Cat #V9879), dissolved in water to 1 mM stock, and used at 100 nM final concentration. Dyngo-4a (AbCam, Cat #ab120689) was dissolved in DMSO to 30 mM, stored protected from light, and added to cells to 30 μM final concentration in serum-free DMEM. Protease inhibitor cocktails were purchased from Roche (Cat #04693159001, 11836170001) and used according to manufacturer recommendations. Phosphodiesterase inhibitors, IBMX (3-Isobutyl-1-Methylxanthine) purchased from Sigma-Aldrich (Cat #I5879) and Rolipram from Tocris (Cat #0905) were dissolved in ethanol to make 100 mM and 10 mM stocks, respectively. Alexa 647-conjugated mouse anti-myc antibody (Cell Signaling, Cat #2233S) was used at 1:50. Rabbit anti-phospho-PKA substrate (100G7E) antibody was purchased from Cell Signaling (Cat #9624) and used at 1:1000. Mouse anti-GAPDH antibody was purchased from Millipore (Cat #MAB374) and used at 1:1000. Secondary IRDye antibodies were purchased from Li-COR Biosciences and used at 1:10,000 in Odyssey Blocking Buffer (Li-COR Biosciences, Cat #927-50000).
3
Platelet Modulation in Colorectal Cancer
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Two different platelet treatments were performed: (A) washed platelets (20,000,000-30,000,000) were incubated for 5 min in the absence or presence of compounds (final concentration: 100µM 3-BP, 10 µM CinnGEL, 100 µM NSC87887). Subsequently, platelet agonist collagen (2 µg/mL) was added to the samples and after 10 min, the platelets were collected, washed and lysed as described below; (B) Platelets collected from co-cultures with colorectal cancer cells were washed with NaCl 0.9% and lysed in 2× concentrated Laemmli buffer (100 mM Tris-HCl [pH 6.8], 200 mM dithiothreitol, 4% SDS, 0.1% bromophenol blue and 20% glycerol) and samples were boiled for 10 min. Cell extracts were resolved by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to polyvinylidene difluoride membranes (Merck chemicals BV, Darmstadt, Germany). Membranes were blocked in 50% odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) in TBS and incubated overnight at 4 °C with a primary antibody. After washing in TBS-T (TBS with 0.5% Tween 20), membranes were incubated with IRDye antibodies (LI-COR Biosciences, Lincoln, NE, USA) for 1 h. Detection was performed using Odyssey reader and analyzed using the manufacturer’s software. For the primary antibodies used, see Supplementary Table S1 .
4
Quantification of FLAG-tagged Proteins in Yeast
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Overnight cultures were grown from single S. cerevisiae colonies in 3 ml of YEPD at 30°C in roller drums. The saturated cultures were diluted 100-fold into 3 ml of fresh YEPD medium and grown for 5 h at 30°C in roller drums. The cultures were quickly chilled in an ice-water bath and spun down in 1.5-ml tubes. The cell pellet was washed in 500 μl of water and incubated in 200 μl of 0.1 M NaOH for 5 min at room temperature. The pellet was resuspended in 50 μl of 1× Laemmli buffer, and western blots were performed using standard molecular biology procedures [76 ]. The anti-FLAG antibody (F1804, Sigma) and the anti-H3 antibody (ab1791, Abcam) were visualized using IRDye antibodies (926-68072 and 926-32211, Licor) on an Odyssey imager. The raw signal in each lane was quantified using ImageJ using the rectangle-select tool followed by the Analyze-Measure tool. For each lane, the FLAG signal was divided by the H3 signal and normalized to a maximum of 10 within each blot. Uncropped images of blots and their quantification are provided in https://github.com/rasilab/ribosome_collisions_yeast/tree/master/data/western . The normalization of lanes is done in https://github.com/rasilab/ribosome_collisions_yeast/blob/master/analysis/western/western_analysis.md .
5
Western Blot Analysis of Protein Samples
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Subconfluent cells were lysed on ice in 2x concentrated Laemmli buffer (100 mM Tris–HCl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.1% bromophenol blue and 20% glycerol) and samples were boiled for 10 min. Cell extracts were resolved by SDS–PAGE and transferred to polyvinylidene difluoride membranes (Merck chemicals BV, Amsterdam, the Netherlands). Membranes were blocked in 50% odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) in PBS/0.05% Tween-20 and incubated overnight at 4°C with primary antibody. After washing in PBS-T, membranes were incubated with IRDye® antibodies (LI-COR Biosciences, Lincoln, NE) for 1 h. Detection was performed using Odyssey reader and analyzed using manufacturers software. For antibodies used see Supplementary Table 1 .
6
Protein Extraction and Western Blot Analysis
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Cells were lysed on ice in RIPA buffer (Thermo Scientific, J62524) containing protease inhibitor (Thermo Scientific, Pierce A32965) and phosphatase inhibitor (Thermo Scientific, Pierce A32957). Lysed cells were spun down and supernatant was incubated with NuPAGE™ LDS sample buffer (Invitrogen, NP0007) for 5 minutes at 95 °C. Proteins were separated using NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels (Thermo Fisher Scientific, NP0321BOX) in NuPAGE™ MES SDS Running Buffer (Thermo Fisher Scientific NP0002), followed by transfer onto nitrocellulose membranes (BIO RAD, 1620115) using Towbin Buffer (2.5 mM Tris, 19.2 mM glycine, pH 8.3) containing 20% methanol. Membranes were blocked with blocking buffer (3% milk, TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5)) for 30 minutes at room temperature, followed by incubation with primary antibody in 1% milk and in TBST buffer overnight (16 hour) at 4 °C. Membrane was washed 3 times in TBST buffer for 10 minutes and incubated with secondary IRDye antibodies (1:5000 LI-COR) in blocking buffer (1% milk, TBST, 0.001% SDS) for 30 minutes at room temperature. Membranes were washed 3 times in TBST buffer and scanned on an Odyssey CLx Imaging System (LI-COR). The following antibodies were used: ZAG (Santa Cruz, sc-13585 1:200), P-Histone H3 (Cell signaling, 34655 1:1000) IRDye 680 RD (LiCor, 926-68072 1:2000).
7
Autophagy Regulation by ATG16L1
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For turnover assays, ATG16L1 rs2241880 knock-in AGS cell lines were subjected to serum starvation (Earle’s Balanced Salt Solution (Sigma) supplemented with sodium bicarbonate 2.2 g/L) or chloroquine (40uM) (Sigma) treatment, for 6 hours.
Western blot was performed as described previously.64 (link) After incubation with the primary antibodies (GRP78 #3177S, LC3B-I/II #2775S, p62 #5114S; Cell Signaling Technology), membranes were incubated with IRDye antibodies (LI-COR Biosciences, Lincoln, NE, USA) or Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (BioRad). Detection was performed using the Odyssey reader or the ImageQuant LAS 500 (GE Life Sciences, Uppsala, Sweden). See supplement for details.
Western blot was performed as described previously.64 (link) After incubation with the primary antibodies (GRP78 #3177S, LC3B-I/II #2775S, p62 #5114S; Cell Signaling Technology), membranes were incubated with IRDye antibodies (LI-COR Biosciences, Lincoln, NE, USA) or Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (BioRad). Detection was performed using the Odyssey reader or the ImageQuant LAS 500 (GE Life Sciences, Uppsala, Sweden). See supplement for details.
8
Exosome Protein Isolation and Analysis
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Following incubation with exosomes for 24 h, cells and isolated exosomes were collected, washed with DPBS and lysed in radio-immunoprecipitation assay (RIPA) buffer with Halt protease and phosphatase inhibitors (all from Thermo Scientific). Protein concentration was determined using the Pierce BCA Protein Assay (Thermo Scientific). Equivalent amounts of protein lysates were loaded on to 4–20% Mini-PROTEAN® TGX gels (Bio-Rad) and transferred to EMD Millipore Immobilon™-FL PVDF (Fisher Scientific). Primary antibodies CD63 (HPA010088, Sigma), β-tubulin (T5201, Sigma), cyclophilin-B (43603S, Cell Signaling) and -actin (3700S Cell Signaling) were used for detection. Secondary IRDye antibodies (LI-COR) were used together with the Odyssey® Fc Imaging System (LI-COR).
9
Quantifying MMP2 and MMP9 Levels
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Tissue explants and isolated EC were cultured and treated with or without rGROα in the presence of EGM-2-MV under the same conditions as described above for the respective angiogenesis assays. Conditioned media was collected after 4d and 8 h, respectively, centrifuged for 10 min at 800 × g to remove cell debris and stored at −80°C before analysis. Proteins were resolved on 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, blocked with Odyssey block (LI-COR Biosciences) and incubated with antibodies against MMP2 or MMP9 (Cell Signalling, catalogue #4022 and #3852, respectively). Secondary IRDye antibodies were obtained from LI-COR (catalogue #926-68,020 and #926-68,073). Detection and quantification of band intensity was performed using Odyssey Infrared Imaging System and Image Studio analysis software (version 3.1.4).
10
Caspase-3 Protein Analysis in Gut Chips
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For the caspase Western blotting studies, cells cultured in the gut chips were trypsinized, pelleted, extracted for total protein with RIPA buffer, run on SDS-PAGE, transferred to nitrocellulose, and Western blots were performed with antibodies to caspase-3 (Cell Signaling Technology, Danvers, MA) and actin (Developmental Studies Hybridoma Bank, Iowa City, IA) as previously described [16 (link)]. Secondary IRDye antibodies were purchased from Li-Cor Biosciences (Lincoln, NE) and the blot was scanned on the Li-Cor Odyssey scanner.
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