Whole-cell extracts were prepared in ice-cold lysing buffer (1 ml PBS) was added with 10 µl Triton X-100, 10 µl SDS 10%, 5 µl DTT 1 M, 6 µl PMSF 0.1%, 10 µl aprotinin). Total proteins (50 µg) were separated by electrophoresis in 10% denaturing SDS/polyacrylamide gel, then transferred to Hybond ECL nitrocellulose membrane (GE Healthcare Europe, Milan, Italy). After saturation of non-specific binding sites with 5% non-fat milk in Tris-buffered saline (TBS) 1x-Tween 20 0.05%, the membrane was immunoblotted overnight at 4°C with the primary antibody against COX-2 (1∶250) and subsequently probed with an anti-goat secondary antibody (1∶1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C. The membrane was stripped (Restore Western Blot Stripping buffer, Pierce Biotechnology, Rockford, IL, USA) and re-immunoblotted with anti-actin primary antibody (1∶10000) and then with anti-rabbit secondary antibodies (1∶7500) (Santa Cruz Biotechnology Inc.). The immunoreactive bands were visualized through enhanced chemiluminescence using the ECL-plus kit (GE Healthcare Europe) following the manufacturer's protocol. The bands were quantified by densitometric analysis using Image J 1.43 software. The results were evaluated as relative units determined by normalization of the density of each band to that of the corresponding actin protein band.
Anti goat secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The anti-goat secondary antibody is a laboratory reagent used in immunoassay techniques, such as Western blotting and immunohistochemistry. It is designed to detect and bind to primary antibodies raised against goat antigens, thereby amplifying the signal and enabling the visualization of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Ecl plus kit, by GE Healthcare (2 mentions)
Anti actin primary antibody, by Santa Cruz Biotechnology (2 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti erk2, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase hrp coupled secondary anti mouse and anti rabbit antibodies, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Goat anti type1 collagen antibody, by Southern Biotech (2 mentions)
His tag antibody, by Abcam (1 mentions)
Trps1 antibody, by R&D Systems (1 mentions)
Anti mouse secondary antibody, by Proteintech (1 mentions)
β actin antibody, by Proteintech (1 mentions)
Anti α tubulin b 7, by Santa Cruz Biotechnology (1 mentions)
Anti c kit, by R&D Systems (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
12 well plate, by Sarstedt (1 mentions)
Sc 34760, by Santa Cruz Biotechnology (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
16 protocols using anti goat secondary antibody
1
Evaluation of COX-2 Expression
2
Western Blot Antibody Optimization
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CHD4 and his-tag antibodies were acquired from Abcam. The TRPS1 antibody was purchased from R&D Systems. β-Actin antibody and anti-mouse secondary antibody were purchased from Proteintech. The ΔNp63 antibody was bought from Abways Technology. Anti-rabbit secondary antibody and anti-goat secondary antibody were purchased from Santa Cruz Biotechnology.
For western blotting, protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and immunoblotted with antibodies. Blots were developed with enhanced chemiluminescence western blotting reagent (Pierce/Thermo Scientific).
For western blotting, protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and immunoblotted with antibodies. Blots were developed with enhanced chemiluminescence western blotting reagent (Pierce/Thermo Scientific).
3
Protein Extraction and Western Blot Analysis
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Protein extracts were prepared by lysing germ cells isolated as describe above (“RNA-seq” section) or K562 cells in RIPA Lysis buffer (Millipore) containing 50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 500 mM NaCl, 1 mM EDTA, and 1× protease inhibitor cocktail (Roche Applied Science). Protein samples were resolved by SDS–polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with the indicated antibodies. Detections were performed using ECL reagents (Pierce). Kit protein was detected with 1:2000 anti-c-kit (R&D Systems AF1356) and 1:2000 of anti-goat secondary antibody (Santa Cruz sc2020). CTCF protein was detected with anti-CTCF B-5 (Santa Cruz sc-271514) and 1:2000 of an anti-mouse secondary antibody (Santa Cruz sc-2005). Tubulin was detected using 1:5000 anti-α Tubulin B-7 (Santa Cruz sc-5286) and 1:2000 of an anti-mouse secondary antibody (Santa Cruz sc-2005). Images were quantified using the ImageJ (v1.48) software and normalized to Tubulin.
4
Western Blot Analysis of TLR4 Expression
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Whole-cell extracts were prepared in ice-cold lysing buffer (50 mm Tris–HCl, pH 7.4, 50 mm NaCl, NP-40 1%, Triton X-100 1%, 0.5 mm EDTA, sodium deoxycholate 1%) containing protease inhibitors (5 mm DTT, PMSF 0.1%, aprotinin 1%). Total proteins (65 μg) were separated by electrophoresis in 8% denaturing SDS/polyacrylamide gel and then transferred to Hybond ECL nitrocellulose membrane (GE Healthcare Europe, Milan, Italy). After saturation of nonspecific binding sites with 5% nonfat milk in Tris-buffered saline (TBS) 1x-Tween 20 0.05%, the membrane was immunoblotted overnight at 4 °C with the primary antibody against TLR4 (1:500) and subsequently probed with an anti-goat secondary antibody (1:1000) (Santa Cruz Biotechnology Inc.,) overnight at 4 °C. The membrane was stripped (Restore Western Blot Stripping buffer, Pierce Biotechnology, Rockford, IL, USA) and re-immunoblotted with anti-actin primary antibody (1:7500) and then with anti-rabbit secondary antibodies (1:5000) (Santa Cruz Biotechnology Inc.). The immunoreactive bands were visualized through enhanced chemiluminescence using the ECL-plus kit (GE Healthcare Europe) following the manufacturer’s protocol.
5
Quantifying ApoD Expression in Osteoblasts
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MC3T3-E1 cells were grown to confluence in 12-well plates (Sarstedt), cultured in control or osteogenic medium for 21 days and analyzed for ApoD expression and secretion on days 0, 7, 14 and 21. Twenty four h prior to protein or RNA extraction, medium was changed for FBS-free MEM which was collected at time of extraction to recuperate secreted ApoD. Cellular proteins were extracted in lysis buffer for immunoblotting, as RNA was retrieved by TriZol (Invitrogen) for PCR analysis. For protein secretion, 40 μL of media was loaded per well; for protein expression, 10 μg of cell extract was loaded per well. All samples were mixed with 5 × denaturing loading buffer and resolved on a 12% SDS-PAGE (Bio-Rad Laboratories, Mississauga, Canada). Gels were transferred onto PVDF membranes and immunoblotted with a mouse-specific goat anti-ApoD (1:1000, sc34760, Santa- Cruz sc34760 biotechnology, Dallas, TX, USA), followed by an anti-goat secondary antibody (1:10,000, Santa Cruz) coupled to HRP. For ApoD secretion, membranes were dyed with Amido Black as a loading reference; for ApoD expression, membranes were incubation with a mouse monoclonal anti-β-actin (1:5000, Sigma-Aldrich). Proteins were revealed with chemiluminescence (ECL, GE Healthcare, Quebec, Canada) using the Fusion FX7 (Vilber Lourmat, France) apparatus and software.
6
Ventral Prostate Tissue Analysis
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Samples of the ventral prostate obtained from five animals in each group were frozen, weighed, and homogenized in 50 mL/mg of lysis buffer. The technique used has been previously briefly described by Fávaro and Cagnon [27 (link)]. In brief, tissue slices were incubated with primary antibodies diluted in 1% BSA at 4 °C overnight. The following primary antibodies were used: anti-CD36 (L-17, Santa Cruz Biotechnology sc-5523), anti-SCAP (9D5, Santa Cruz Biotechnology sc-13,553), anti-LIMPII (D-4, Santa Cruz Biotechnology sc-55,571), anti-SREBP-1 (H-160, Santa Cruz Biotechnology sc-8984), and anti-caspase-3 (Abcam ab4051) at a dilutions of 1:500, 1:250, 1:250, 1:500, respectively; anti-β-actin (Abcam ab8227) was used as a control. An anti-goat secondary antibody (Santa Cruz Biotechnology sc-2354) was used against all primary antibodies at a 1:1000 dilution. The results are expressed as the mean of the ratio between the intensity of each band against the intensity of the β-actin band.
7
Western Blot Analysis of Mouse Liver and Serum
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Mouse serum (4 μl) was diluted with 12 μl of PBS, and mouse liver (50 mg) was homogenated using 125 μl of homogenate buffer (20 mM Tris-HCl (pH 7.4), 0.1% SDS, 0.1% Triton X-100, 0.01% sodium deoxycholate and 1 × Complete protease inhibitor cocktail (Roche Diagnostics)). Samples (16 μl) were mixed with 4 μl of Laemmli sample buffer (Bio-Rad, Hercules, CA) and then denatured at 95 °C for 2 min. Total proteins were separated by electrophoresis on a 5–20% gradient polyacrylamide gel (ATTO Corporation, Tokyo, Japan) and transferred onto polyvinylidene difluoride membranes. Blots were probed with goat primary antibodies against Apolipoprotein A-I (1:500, sc-23605, Santa Cruz Biotechnology, Santa Cruz, CA) and Apolipoprotein B (1:500, sc-11795, Santa Cruz Biotechnology), or Scavenging receptor SRB1 (1:2,000, ab52629, Abcam, Cambridge, UK) and β-actin (1:500, ab6276, Abcam), and then incubated with anti-goat secondary antibody (1:2,000, sc-2020, Santa Cruz Biotechnology), anti-rabbit secondary antibody (1:2,000, sc-2004, Santa Cruz Biotechnology) or anti-mouse secondary antibody (1:2,000, sc-2005, Santa Cruz Biotechnology) conjugated with horseradish peroxidase. Blots were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA) and analysed by a ChemiDoc System (Bio-Rad).
8
Antibody Reagents for Septin-Based Research
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Anti-SEPT7 antibodies were obtained from IBL international (#JP18991), rabbit anti-anillin antibodies were reported earlier40 (link). Other antibodies used were SEPT2 (#11397-1-AP, Acris), SEPT9 (#10769-1-AP, Acris), SEPT6 (sc-20180, Santa Cruz Biotech), SEPT8 (sc-48937, Santa Cruz Biotech), EF2 (sc-13004-R, Santa Cruz Biotech), GAPDH (#MAB374, Millipore), tubulin-α (T6199, Sigma), GFP (sc-9996, Santa Cruz Biotech), GST (sc-138, Santa Cruz Biotech), FLAG (#200472-21, Stratagene) and p38 MAPK (#9212, Cell Signaling Technology). All Alexa-dye labeled secondary antibodies and Alexa fluor-647-conjugated phalloidin (#A22287) were from Invitrogen. HRP-labeled anti-mouse (#115-035-003) and anti-rabbit (#111-035-003) secondary antibodies were from Dianova; anti-goat secondary antibody was from Santa Cruz Biotech (sc-2033). DAPI for DNA staining was from Carl Roth (#6335.1). Polybrene (H9268), doxycycline (D9891) were from Sigma.
9
Western Blot Analysis of Protein Markers
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Cells were lysed in ice-cold 1X RIPA lysis buffer (Santa Cruz Biotechnology, Inc., CA, USA) containing: PMSF, sodium orthovanadate and protease inhibitor cocktail. After incubation in ice-cold buffer for 30 min, the cell lysate was centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was harvested. The detailed methods of the Western blot procedure were performed as previously described (Gao et al., 2003 (link)). The intensity of the chemiluminescence signal was normalized to that of β-actin as indicated. The following antibodies and the respective dilutions were used: 1:2,000 for β-actin, 1:2,000 for VCAM-1, 1:1,000 for cleaved caspase-3 and 1:2,000 for anti-rabbit secondary antibody (Cell Signaling Inc., Danvers, MA, USA); 1:1,000 for tyrosine hydroxylase (TH), 1:1,000 for p-Akt, 1:1,000 for ICAM-1 and 1:2,000 for anti-goat secondary antibody (Santa Cruz Biotechnology Inc.). The TATA box binding protein (TBP) was the nuclear protein loading control for p65. The antibody for TBP was from ABCAM and was used at 1:1,000 dilution.
10
Immunoblotting of Phosphorylated Proteins
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Lipofectamine 2000 and horseradish peroxidase (HRP)-coupled secondary anti-mouse and anti-rabbit antibodies were from ThermoFisher Scientific (Waltham, MA). Cycloheximide and mouse anti-FLAG (M2) antibody were from Sigma-Aldrich (St Louis, MI). FLT3 ligand (FL) was from ORF Genetics (Reykjavik, Iceland). Rabbit anti-phospho-AKT (pSer473), mouse anti-phosphotyrosine (4G10), mouse anti-mono-ubiquitin antibodies were from Abcam (Cambridge, UK), Merck Millipore (Billerica, MA) and Covance Research Products (Princeton, NJ), respectively. Rabbit anti-phospho-ERK1/2 (pThr202/pThr204), goat anti-AKT, rabbit anti-ERK2 and anti-goat secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The phycoerythrin-labeled anti-FLT3, mouse anti-phospho-p38 and anti-p38 antibodies were from BD Transduction Laboratories (Franklin Lakes, NJ). Rabbit anti-FLT3 antibody was described previously [29 (link)].
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