Western immunoblotting analysis was performed using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques to determine levels of p-mTOR rabbit polyclonal antibody (#2971, dilution 1:1000), mTOR rabbit polyclonal antibody (#2972, dilution 1:1000), p-STAT3 (Tyr705)rabbit monoclonal antibody (#9145, dilution1:1000), p-STAT3 (Ser727)rabbit polyclonal antibody (#9134, dilution 1:1000), STAT3 rabbit monoclonal antibody (#12640, dilution 1:1000), p-AMPKα (T172)rabbit monoclonal antibody (#2535, dilution 1:1000), AMPKαrabbit polyclonal antibody (#2532, dilution 1:1000), p-JAK2rabbit monoclonal antibody (#3776, dilution 1:1000), p-4E-BP1 Rabbit monoclonal antibody (#2855, dilution 1:1000) (Cell Signaling Technology),LC3-I and -II rabbit polyclonal Ab (NB100-2220, dilution 1:1000) (Novus Biologicals), β-Actin goat polyclonal antibody (sc-1616, dilution 1:1000), anti-p62 rabbit polyclonal antibody (sc-25523, dilution 1:500) (Santa Cruz Biotechnology). Incubation of infrared dye-labeled secondary antibodies (LI-COR Biotechnology) was performed in darkness for 1 h at room temperature. After extensive washing, membranes were scanned and analyzed on an Odyssey™ scanner (LI-COR Biotechnology).
Infrared dye labeled secondary antibody
Manufactured by LI COR
Sourced in Germany
Infrared dye-labeled secondary antibodies are specialized laboratory reagents designed for use in various immunoassay techniques. They consist of secondary antibodies that have been conjugated with infrared fluorescent dyes. These dyes enable detection and quantification of target proteins or molecules using infrared imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (4 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Odyssey infrared imager, by LI COR (2 mentions)
P 4ebp1, by Cell Signaling Technology (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Licor scanner, by LI COR (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Rabbit anti caspase 9, by Cell Signaling Technology (1 mentions)
Rabbit anti bim, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Rabbit histone h3, by Abcam (1 mentions)
Goat anti rabbit igg conjugated to horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Mouse β actin, by Merck Group (1 mentions)
Mouse anti bax, by BD (1 mentions)
Mouse anti parp, by Cell Signaling Technology (1 mentions)
Mouse anti caspase 8, by Alexis Biochemicals (1 mentions)
Mouse anti noxa, by Alexis Biochemicals (1 mentions)
Rat anti bmf, by Alexis Biochemicals (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
12 protocols using infrared dye labeled secondary antibody
1
Quantitative Western Blot Analysis of Signaling Pathways
2
Tau Monomer Denaturation and Dot Blot
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T40 tau monomer or AD-tau were chemically and thermally denatured by 1:10 dilution in 8 M guanidine hydrochloride and heating at 100 °C for 15 min, and non-denatured controls were diluted in Tris-buffered saline (TBS) at RT. Denatured or non-denatured tau was then diluted 1:50 in TBS and applied to 0.2 μm nitrocellulose membrane using a vacuum apparatus. Each dot of recombinant tau monomer was loaded with 0.25 μg tau and AD-tau was titrated to determine equivalent loading based on detection of total tau with K9JA antibody. For western blots, tau isoforms and fragments were diluted in SDS sample buffer, heated 10 min at 100 °C, run on 12.5% SDS-PAGE gels and transferred to 0.2 μm nitrocellulose membrane. Immunoblots and dot blots were probed with either total tau control antibody K9JA diluted to 2 μg/mL or conformation-selective mAbs diluted in 5% non-fat milk at 20 μg/mL at 4 °C. Infrared dye labeled secondary antibodies (LiCor) were used to detect primary antibody binding, with analysis on a LiCor scanner (LiCor).
3
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed as described previously [28 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-Noxa, rat anti-Bmf (Alexis Biochemicals, Grünberg, Germany), mouse anti-Bcl-2, rabbit anti-Bcl-xL, mouse anti-Bax, rabbit anti-Bak (BD Transduction Laboratories, Heidelberg, Germany), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-Bim, mouse anti-PARP (Cell Signaling, Beverly, MA), acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-Mcl-1 (Stressgene, Victoria, BC), rabbit histone H3 (Abcam, Cambridge, UK). Mouse anti-GAPDH (HyTest, Turku, Finland), mouse α-Tubulin (Calbiochem, Darmstadt, Germany) or mouse β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) were used as secondary antibodies. Enhanced chemiluminescence (Amersham Bioscience, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odyssey Imaging System, LICOR Bioscience, Bad Homburg, Germany) were used for detection. Representative blots of at least two independent experiments are shown.
4
Western Blot Protein Analysis Protocol
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Cell pellets were lysed in RIPA lysis buffer and the protein content was determined using a DC protein assay kit (Bio-Rad Laboratories). The proteins were run on electrophoresis gels and transferred to nitrocellulose membranes (GE Osmonics Labstore) as described previously [50 (link)]. Membranes were blocked for 1 hr in blocking buffer, incubated with primary antibodies against XIAP (BD Biosciences, 610762; as per company, this antibody detects two bands and the lower band is specific for XIAP, as indicated by arrows in the figures); cIAP1 (Abcam, ab2399); cIAP2 (Epitomics, S2700; as per company, this antibody detects two bands and the upper band is specific for cIAP2, as indicated by arrows in the figures); Smac (BD Biosciences, 612246); caspase 3, (Cell Signaling, 9665) caspase 8 (Cell Signaling 9746), and caspase 9 (Cell Signaling, 9502); polyclonal antibody to Mcl-1 or Bcl-xL; and mouse monoclonal antibody to Bcl-2 (Santa Cruz, CA) or to GAPDH (Abcam, Cambridge, MA). The antibody to poly(ADP-ribose) polymerase was from BIOMOL International (Plymouth Meeting, PA). Following washing with PBST, membranes were incubated for 1 hr with infrared dye-labeled secondary antibodies (LI-COR Biosciences), scanned, and visualized using a LI-COR Odyssey infrared imager.
5
Western Blot Analysis of DNA Damage Signaling
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Cell pellets were lysed in RIPA lysis buffer and the protein content was determined using a DC protein assay kit (Bio-Rad Laboratories). The proteins were run on electrophoresis gels and transferred to nitrocellulose membranes (GE Osmonics Labstore) as described previously. [24 (link)] Membranes were blocked for 1 hour in blocking buffer, incubated with primary antibodies against total ATM (ab17995; Abcam, Cambridge, MA), phospho-ATMSer1981 (EMD Millipore, Billerica, MA), total p53 (EMD Millipore, Billerica, MA), phospho-p53Ser15 (05-740; Cell Signaling Technology, Danvers, MA), CHK-2 (3440) and phospho-CHK-2Thr68 (2661; Cell Signaling Technology, Danvers, MA), MCL-1 (AHO0102; Life Technologies Corporation, Carlsbad, CA), PUMA (3043; ProSci Incorporated, Poway CA), BAX (sc-20067; Santa Cruz Inc, Dallas, TX) and GAPDH (5174; Cell Signaling Technology, Danvers, MA).
Following washing with PBST, membranes were incubated for 1 hour with infrared dye-labeled secondary antibodies (LI-COR Biosciences), scanned, and visualized using a LI-COR Odyssey infrared imager.
Following washing with PBST, membranes were incubated for 1 hour with infrared dye-labeled secondary antibodies (LI-COR Biosciences), scanned, and visualized using a LI-COR Odyssey infrared imager.
6
Western Blot Analysis of SARS-CoV-2 Nucleocapsid
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Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% skim milk in PBS-T overnight at 4°C, followed by incubation with a primary antibody for 2 h at room temperature. After three washes with PBS-T, the membranes were subsequently incubated with the appropriate infrared dye-labeled secondary antibodies (Li-Cor Biosciences) for 1 h at room temperature, followed by five washes with PBS-T before being scanned with Odyssey CLx imaging system (Li-Cor Biosciences). Rabbit anti-SARS-CoV-2 N was generated in-house as described above; Flag tag and β-actin antibodies were purchased from Cell Signaling Technology. The secondary antibodies IRDye 680 goat anti-rabbit IgG and IRDye 800 CW goat anti-mouse IgG were purchased from Li-Cor Biosciences.
7
Phagocytosis Assay with pHrodo Conjugates
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pHrodo E. coli (K12 strain) bioparticle conjugates (catalog no: P35366 for green florescence, and P35361 for red florescence) and an opsonizing reagent (catalog no: E2870) were purchased from Thermo Fisher Scientific (Carlsbad, CA). Cell culture media were purchased from Thermo Fisher Scientific. The antibodies for Western blotting included anti-ARP2 (catalog no: 5614), anti-p-cofilin (catalog no: 3313), anti-total cofilin (catalog no: 5175), anti-βPIX (catalog no: 4515), anti-mouse p-STAT3 (Tyr705, catalog no: 9131), and anti-total STAT3 (catalog no: 9139); these were obtained from Cell Signaling Technologies (Danvers, MA). Anti-β-actin antibodies (clone AC-15, catalog no: A5441), an anti-Rac1 magnetic bead conjugate (catalog no: 16-319), and anti-Rac1 antibodies (catalog no: 05-389) were purchased from Millipore-Sigma (St. Louis, MO). A Rac1 activation magnetic bead pulldown assay kit (catalog no: 17-10394) was purchased from Millipore-Sigma (St. Louis, MO). Infrared dye-labeled secondary antibodies were obtained from Li-Cor Biosciences (Lincoln, NE). The STAT3 inhibitor stattic (catalog no: sc-202818) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Rac1 inhibitor NSC 23766 (catalog no: 13196) was purchased from Cayman Chemicals (Ann Arbor, MI). A MemBrite cell surface staining kit was purchased from Biotium (catalog no: 30093-T, Fremont, CA).
8
MDA-MB-231 Protein Expression Analysis
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MDA-MB-231 cells were lysed on ice using RIPA buffer (cat. no. R0278; Sigma-Aldrich; Merck KGaA). Protein concentration was evaluated using the BCA method. Proteins (30 µg) were separated by 10% SDS PAGE and transferred onto PVDF membranes. After blocking for 1 h in 5% skimmed milk dissolved in PBS, membranes were incubated with primary antibodies against cyclin E (cat. no. SC248 1:500 Santa Cruz Biotechnology, Inc.), PARP (cat. no. AY 0276; 1:1,000; Abways Technology, Inc.), cleaved PARP (cat. no. CY5035; 1:500; Abways Technology, Inc.) and β-actin (cat. no. A5441; 1:5,000; Sigma-Aldrich; Merck KGaA) at 4°C overnight. The membranes were washed three times with PBST (0.1% Tween) and incubated with infrared dye-labeled secondary antibody (LI-COR Biosciences) for 1 h at room temperature. The signal on the membrane was visualized using LI-COR Odyssey System.
9
Immunoprecipitation and Western Blotting
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Cell lysates in RIPA buffer were immunoprecipitated with an anti-Flag antibody (Sigma-Aldrich) using the SureBeads magnetic beads system (Bio-Rad). Precipitated proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The blots were blocked in 5% skim milk in PBS for 1 hour and incubated with the anti-Flag antibody overnight at 4°C. After washing with PBS, the blots were incubated with an infrared dye-labeled secondary antibody (Li-Cor Biosciences) for 1 hour at room temperature and then washed again. The blot was scanned using Odyssey Infrared Imaging System (Li-Cor Biosciences).
10
Cytoplasmic and Nuclear Protein Extraction
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