Whole cell lysates were collected in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40). Isolation of cytosolic and nuclear fractions was done with an NE-PER kit (Pierce; Rockford, IL) following the manufacturer’s protocol. Protein (30 μg) was resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted overnight at 4 °C with primary antibodies. The various primary antibodies used were CTD110.6 (IgM, UAB), GLI1 (Cell Signaling; 2643), GLI2 (Boster Biological Technology; Pleasanton, CA; PA1941), Histone H3 (Cell Signaling; 4499), OGA (Santa Cruz Biotechnology; Dallas, TX; sc-376429), OGT (Sigma-Aldrich; O6264), PKM2 (Cell Signaling; 4053), phospho-Y105–PKM2 (Cell Signaling; 3827), and RL2 (Abcam; Cambridge, MA; ab2739). Alpha-tubulin (Cell Signaling; 12351) or beta-actin (Sigma-Aldrich; A3854) was used to confirm equal loading. Anti-rabbit or anti-mouse HRP-conjugated secondary antibody (GE Healthcare; Chicago, IL) was used for detection, and blots were developed with either ECL or SuperSignal substrate (Pierce) and imaged on films or Amersham Imager 600. The purity of cytosolic and nuclear fractions was confirmed with beta-tubulin (2146; Cell Signaling) or histone deacetylase 1 (Cell Signaling; 2062) antibodies, respectively. The depicted images are representative of experiments repeated at least three times.
Anti rabbit or anti mouse hrp conjugated secondary antibody
Manufactured by GE Healthcare
Sourced in United States
Anti-rabbit or anti-mouse HRP-conjugated secondary antibodies are laboratory reagents used for the detection and visualization of target proteins in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. These secondary antibodies are specifically designed to bind to the primary antibodies raised against rabbit or mouse antigens, and they are conjugated with the enzyme horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
α tubulin, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Histone deacetylase 1, by Cell Signaling Technology (2 mentions)
β tubulin, by Cell Signaling Technology (2 mentions)
Phospho y105 pkm2, by Cell Signaling Technology (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Amersham imager 600, by Cytiva (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence system, by GE Healthcare (2 mentions)
Phosstop, by Roche (2 mentions)
Complete mini, by Roche (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Imager 600, by Cytiva (1 mentions)
β actin a3854, by Merck Group (1 mentions)
Hif 1α, by BD (1 mentions)
Anti ac tub, by Merck Group (1 mentions)
Chemidoc scanning system, by Bio-Rad (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
8 protocols using anti rabbit or anti mouse hrp conjugated secondary antibody
1
Subcellular Fractionation and Immunoblotting
2
Subcellular Fractionation and Immunoblotting
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Whole cell lysates were collected in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40). Isolation of cytosolic and nuclear fractions was done with an NE-PER kit (Pierce; Rockford, IL) following the manufacturer’s protocol. Protein (30 μg) was resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted overnight at 4 °C with primary antibodies. The various primary antibodies used were CTD110.6 (IgM, UAB), GLI1 (Cell Signaling; 2643), GLI2 (Boster Biological Technology; Pleasanton, CA; PA1941), Histone H3 (Cell Signaling; 4499), OGA (Santa Cruz Biotechnology; Dallas, TX; sc-376429), OGT (Sigma-Aldrich; O6264), PKM2 (Cell Signaling; 4053), phospho-Y105–PKM2 (Cell Signaling; 3827), and RL2 (Abcam; Cambridge, MA; ab2739). Alpha-tubulin (Cell Signaling; 12351) or beta-actin (Sigma-Aldrich; A3854) was used to confirm equal loading. Anti-rabbit or anti-mouse HRP-conjugated secondary antibody (GE Healthcare; Chicago, IL) was used for detection, and blots were developed with either ECL or SuperSignal substrate (Pierce) and imaged on films or Amersham Imager 600. The purity of cytosolic and nuclear fractions was confirmed with beta-tubulin (2146; Cell Signaling) or histone deacetylase 1 (Cell Signaling; 2062) antibodies, respectively. The depicted images are representative of experiments repeated at least three times.
3
Western Blot Analysis of VHL and HIF-1α
4
Western Blot Analysis of Larval Testes
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To obtain testes extracts for the Western Blot analysis, larval testes were lysed in an ice-cold buffer containing 20 mM Hepes KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 420 mM NaCl, 30 mM NaF, 0.2 mM Na3Vo4, 25 mM β-glycerophosphate, 0.5 M PMSF, 0.1% NP40, 1× protease inhibitor cocktail (Roche, Basel, Switzerland). For immunoblotting, protein samples were resuspended in 1× Laemmli Buffer, run into SDS polyacrilammide gels, and electroblotted on a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in a phosphate buffer containing 390 mM NaH2PO4 and 610 mM Na2HPO4. After blocking with 5% low-fat dry milk, the membrane was probed with appropriate primary antibody. Anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:5000; GE Healthcare, Chicago, IL, USA) were used as secondary antibodies. The blots were developed by the ECL or ECL Plus method (Amersham Biosciences, Little Chalfont, UK) and signals detected with the ChemiDoc scanning system (BioRad). The antibody dilutions were: Anti-DmATPCL (1:500), anti-acTub (Sigma; 1:10,000), anti Ac-Lys (1:1000; Merck-Millipore, Burlington, MA, USA).
5
Microglia Protein Expression Analysis by Western Blot
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Denaturing SDS-PAGE (BioRad) gels were used to separate 8 µg of microglia protein per lane. Proteins were transferred to nitrocellulose, which was subsequently blocked with 5% milk Tris-buffered saline tween-20. Blots were probed for Sema4A, Hft, or beta-actin for 16 hr at 4℃. Anti-Sema4A (1:200, rabbit polyclonal antibody) was purchased from Abcam. Anti-Hft (1:1,000, rabbit monoclonal antibody) was purchased from Cell Signaling (Danvers, MA). Anti-beta-actin (1:3,000, mouse monoclonal antibody) was purchased from Sigma. Corresponding anti-rabbit or anti-mouse HRP-conjugated secondary antibodies were used (1:5,000, GE Amersham, Amersham, UK). Protein was visualized with ECL reagents (Perkin–Elmer, Waltham, MA) on the FujiFilm LAS-3000 System and Image Reader LAS-3000 software. Densitometry quantification was performed using MultiGauge software.
6
Western Blot Analysis of Retinal Proteins
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Retinal tissue was homogenized in RIPA buffer (Millipore, Billerica, MA, United States) containing protease inhibitor (Complete Mini) and phosphatase inhibitors (phosSTOP, Roche Applied Science, Indianapolis, IN, United States). Protein concentration was measured using the bicinchoninic acid kit (Pierce, Thermo Scientific) according to manufacturer's instructions. Protein samples (15–20 ug) per lane were separated on SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (Millipore, Billerica, MA, United States), blocked in 5% dry milk in 1X Tris-buffered saline with 0.5% Tween-20 (TBST). Membranes were incubated in primary antibodies overnight at 4°C with phospho Akt, total Akt, phospho ERK1/2, total ERK1/2, Bcl-xL, BID, (Cell Signaling Technology, Danvers, MA, United States) diluted in 2% BSA, or beta-Actin (Sigma). Further, the membranes were washed with TBST and incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (GE-Healthcare, Piscataway, NJ, United States), washed (TBST) and detected using enhanced chemiluminescence system (GE-Healthcare, Piscataway, NJ, United States). The band intensities were quantified by densitometry using ImageJ software and normalized to loading controls.
7
Western Blot Analysis of Retinal Proteins
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Immunoblotting experiments were performed as previously described [36 (link)]. Whole retinal tissues (from control and diabetic mice) were isolated and homogenized using RIPA buffer (Millipore, Billerica, MA, USA) consisting of protease (Complete Mini) and phosphatase (phosSTOP, Roche Applied Science, Indianapolis, IN, USA) inhibitor cocktails. PierceTM BCA protein assay (Thermo Scientific, Rockford, IL, USA) was used for the estimation of protein concertation. Around 20 ug protein per sample was used for Western blotting analysis. Samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were blocked with non-fat dry milk (5%), incubated overnight in respective primary antibody (Table 1 ) at 4 °C, treated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (GE-Healthcare, Piscataway, NJ, United States), followed by detection using enhanced chemiluminescence system (GE-Healthcare, Piscataway, NJ, USA). Densitometry analysis was using ImageJ software and normalized to loading controls.
8
Immunoblot Analysis of siRNA-Transfected Cos-7 Cells
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For immunoblots 48-72 hours after siRNA transfection, Cos-7 cells were lysed in RIPA buffer (Sigma) supplemented with fresh proteases inhibitors (Sigma Aldrich 11836170001) on ice for 30 minutes.
A centrifugation at 16,000 g for 10 min at 4˚C was performed to remove the insoluble material. Protein concentrations were determined using a Pierce BCA protein assay kit (Life Technologies, 23227) and equal amounts of protein were analyzed by self-casted 7.5 or 15% SDS-PAGE (30-50 µg of proteins per lane).
For immunoblotting, proteins were transferred to nitrocellulose membranes (BioRad) electrophoretically and incubated with the specified primary antibodies (see above), diluted in 5% non-fat dry milk in Tris buffered saline with Tween 20 (TBST). The blots were further incubated with anti-rabbit or anti-mouse HRP conjugated secondary antibodies (GE Healthcare) and visualized using ECL (GE Healthcare). Where required, images of Western blotting were treated for contrast enhancement and densitometric analyses were performed using ImageJ.
A centrifugation at 16,000 g for 10 min at 4˚C was performed to remove the insoluble material. Protein concentrations were determined using a Pierce BCA protein assay kit (Life Technologies, 23227) and equal amounts of protein were analyzed by self-casted 7.5 or 15% SDS-PAGE (30-50 µg of proteins per lane).
For immunoblotting, proteins were transferred to nitrocellulose membranes (BioRad) electrophoretically and incubated with the specified primary antibodies (see above), diluted in 5% non-fat dry milk in Tris buffered saline with Tween 20 (TBST). The blots were further incubated with anti-rabbit or anti-mouse HRP conjugated secondary antibodies (GE Healthcare) and visualized using ECL (GE Healthcare). Where required, images of Western blotting were treated for contrast enhancement and densitometric analyses were performed using ImageJ.
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