Proteins from Differentiated osteoblasts were separated by SDS- polyacrylamide gels and electrotransferred to PVDF membrane. The membranes were blocked with 5% non-fat dry milk in TBS-Twin for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with antisera raised against Runx2, osteonectin and beta-galactosidase in TBS-Twin containing 1% non-fat dry milk. The membranes were washed thrice for 5 min each with TBS-T and probed with appropriate near-Infrared Fluorescent Secondary Antibodies (LI-COR) in a 1% non-fat dry milk in TBS-T at room temperature for 1 h. After washing, the immune complexes comprising of the target proteins were detected using the Odyssey imaging system from LI-COR. The membranes then were stripped and reprobed with GAPDH controls. The band intensity was quantified and protein expression levels were calculated relative to GAPDH from the same membrane.
Near infrared fluorescent secondary antibodies
Manufactured by LI COR
Near-infrared fluorescent secondary antibodies are specialized laboratory reagents used in various immunoassay techniques, such as Western blotting and immunohistochemistry. These antibodies bind to primary antibodies and emit near-infrared fluorescence, allowing for sensitive detection and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (2 mentions)
Complete mini edta free tablet, by Roche (2 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Phosstop, by Roche (2 mentions)
Lowry assay, by Bio-Rad (2 mentions)
Image studio software, by LI COR (2 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Anti ifitm3, by Proteintech (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
Fc imaging system, by LI COR (1 mentions)
Tween 20, by Merck Group (1 mentions)
Mouse monoclonal anti flag clone m2, by Merck Group (1 mentions)
Odyssey fc system, by LI COR (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Agilent seahorse xfp cell energy phenotype test kit, by Agilent Technologies (1 mentions)
Agilent seahorse wave desktop v2, by Agilent Technologies (1 mentions)
Seahorse xfp analyser, by Agilent Technologies (1 mentions)
7 protocols using near infrared fluorescent secondary antibodies
1
Western Blot Analysis of Osteoblast Proteins
2
Protein Expression Analysis by Western Blot
3
SDS-PAGE and Western Blot Analysis
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To prepare cell extracts, cells were lysed in 1× Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM dithiothreitol, 0.02% bromophenol blue). Viruses, VLPs, and lung homogenates were mixed with 5× Laemmli buffer to obtain 1× Laemmli buffer lysates. Samples were run on SDS-PAGE gels and blotted onto nitrocellulose membranes (Hybond ECL; GE Healthcare). All stainings were performed in Tris-buffered saline mixed with 0.5% Tween-20 (Sigma-Aldrich) and 3% powdered milk. The following antibodies were used: mouse monoclonal anti-Flag (clone M2; Sigma-Aldrich), rabbit polyclonal anti-IFITM3 (Proteintech), mouse monoclonal anti–β-actin (Santa Cruz Biotechnology), mouse monoclonal anti–HIV-1 p24 (ab9071; Abcam), mouse monoclonal anti-IAV M1 (HB-64; American Type Culture Collection), rabbit polyclonal anti-nucleoprotein (NP; a kind gift of A. Nieto, Spanish National Center for Biotechnology, Madrid, Spain), mouse monoclonal anti-A/WSN/33 HA (clone H15-B9-22 from Wistar), rabbit polyclonal anti-A/WSN/33, and mouse monoclonal anti-A/Netherlands/602/2009 HA clone 31C2 (Manicassamy et al., 2010 (link)). Secondary antibody staining was performed using near-infrared fluorescent secondary antibodies (Li-Cor) and images were acquired on an Odissey Fc imaging system. Western blot signal intensities were quantified using the Image Studio software (Li-Cor).
4
Western Blot Analysis of Proteins
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Denatured proteins from either mouse retinal samples or 661 W cells were separated by SDS- polyacrylamide gels (4–12% Tricine gels) and electroblotted onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBS with 0.1% Tween 20, incubated overnight with primary antibodies (dilution 1:1000), probed with near-Infrared Fluorescent Secondary Antibodies (LI-COR) (dilution 1:10000), and visualized with an Odyssey imaging system (LI-COR). The band intensities were quantified, and protein expression levels were calculated relative to either actin or GAPDH from the same membrane after stripping.
5
Western Blot Protein Analysis Protocol
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Cells were lysed in a standard Triton X‐100 lysis buffer containing 25 mM Tris‐HCl (pH = 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X‐100, supplemented with protease and phosphatase inhibitors. Lysates were cleared by centrifugation, and protein concentrations were determined by the Bradford protein assay kit (Bio‐Rad). Equal amounts of total proteins were loaded per lane on 10% polyacrylamide gels, and proteins were separated by electrophoresis and blotted on nitrocellulose membranes. Membranes were blocked with 5% non‐fat milk dissolved in dissolved in tris‐buffered saline ‐ tween 20 (TBS‐T) and stained overnight with the primary antibody (listed in supplementary materials ) and subsequently labeled with near‐infrared fluorescent secondary antibodies (LI‐COR). Membranes were developed using the LI‐COR Odyssey FC system. Protein quantifications and image analyses were performed using the Image Studio Software (LI‐COR).
6
Protein Expression Analysis by Western Blot
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Cells were lysed in modified RIPA buffer (0.1% SDS, 1% NP40, 150 mM NaCl, 1% sodium deoxycholate, 25 mM Tris-HCl pH 7.4) containing protease inhibitors (Complete Mini EDTA-free tablets; Roche) and phosphatase inhibitors (PhosSTOP, Roche). Protein concentrations were measured with a Lowry Assay (Bio-Rad), and 20–60 μg of protein was run on 8–10% SDS-PAGE gels. Protein was transferred on to a 0.45 μm nitrocellulose membrane (Bio-Rad) using a wet transfer or with Trans-Blot Turbo system, blocked for 1 h at room temperature in 5% BSA in 1X TBST or Odyssey Blocking buffer, and probed with antibodies diluted in 5% BSA in 1X TBST or Odyssey buffer. Mouse and rabbit near-infrared fluorescent secondary antibodies (LI-COR Biosciences) were added for 1 h at room temperature. The membrane was subsequently washed in 1X TBST, and imaged with an Odyssey CLx imaging system. Analysis was done with FIJI (Schindelin et al., 2012 (link)) by calculating densitometry relative to the loading control.
7
Metabolic Profiling of GSK3β Knockout Cells
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Equal amounts (15 μg) of proteins from different samples were loaded on 8% acrylamide gel and subjected to electrophoresis (120 volts, 90 minutes). Afterwards, proteins were transferred to nitrocellulose membrane (80 volts, 0.4 Amps, 3 hours) (Whatman 10402594, Dassel, Germany) and blocked for 30 minutes using a 5% milk solution. Membranes were then incubated at 4 o C and proteins identified using near-infrared fluorescent secondary antibodies (Li-Cor) and imaged using a Li- Metabolic profiling of HCT116-GSK3β-WT and HCT116-GSK3β-KO cells.
Metabolism profiling was carried out by Agilent Seahorse XFp Cell Energy Phenotype test kit on a Seahorse XFp analyser (Agilent). Protocols were carried out according to manufacturer guidelines and data was analysed using Agilent Seahorse Wave desktop V2.5. Metabolic pathway analysis was performed using Escher (11) in conjunction with the BiGG database (12) .
Metabolism profiling was carried out by Agilent Seahorse XFp Cell Energy Phenotype test kit on a Seahorse XFp analyser (Agilent). Protocols were carried out according to manufacturer guidelines and data was analysed using Agilent Seahorse Wave desktop V2.5. Metabolic pathway analysis was performed using Escher (11) in conjunction with the BiGG database (12) .
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