Samples were denatured in a buffer (60.0 mM Tris pH 6.8, 10% sucrose, 2% SDS, 4% β-mercaptoethanol) and loaded onto a 12% SDS-Polyacrylamide gel. After the run in electrode buffer (50 mM Tris, pH 8.3, 384 mM glycine, 0.1% SDS), the gels were transferred onto PVDF membranes (Millipore, MA, USA) at 0.8 mA/cm2 for 90 minutes. The membranes were preblocked in blocking buffer (PBS, 5% milk powder, 0.5% Tween 20) for 1 hour and then incubated overnight at 4°C with rabbit polyclonal antibody for UCP3 (Millipore, MA, USA, diluted 1:3000 in blocking buffer). Membranes were washed 3 times 12 minutes in PBS/0.5% Tween 20 and 3 times 12 minutes in PBS; and then incubated 1 hour at room temperature with alkaline phosphatase-conjugated secondary antibody (Promega, WI, USA). The membranes were washed as above described, rinsed in distilled water and incubated at room temperature with a chemiluminescent substrate, CDP-Star (Sigma-Aldrich, MO, USA). Data detection was carried out by exposing autoradiography films (Eastman Kodak Company, NY, USA) to the membranes. Quantification of signals was carried out by Un-Scan-It gel software (Silk Scientific, UT, USA). Values were expressed as optical density arbitrary units and were normalized for mg of proteins. Equal loading was verified by Ponceau S staining.
Alkaline phosphatase conjugated secondary antibody
Manufactured by Promega
Sourced in United States
Alkaline phosphatase-conjugated secondary antibodies are lab equipment used in immunoassays. They function as detection reagents, binding to primary antibodies and catalyzing a colorimetric or chemiluminescent reaction to signal the presence of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Anti pgk1, by Thermo Fisher Scientific (2 mentions)
Protein a sepharose, by GE Healthcare (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Autoradiography film, by Kodak (1 mentions)
Cdp star, by Merck Group (1 mentions)
Un scan it gel software, by Silk Scientific (1 mentions)
Immune star ap substrate, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Multi beads shocker, by Yasui Kikai (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Nitroblue tetrazolium, by Merck Group (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab5168, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Sc 7273, by Santa Cruz Biotechnology (1 mentions)
Sc 9154, by Santa Cruz Biotechnology (1 mentions)
Anti cd36, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
13 protocols using alkaline phosphatase conjugated secondary antibody
1
Western Blot Analysis of UCP3
2
Quantification of P-glycoprotein in Cell Lines
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Total protein concentration for standardized plasma membrane preparations of Madin-Darby canine kidney (MDCK) and multidrug-resistant (MDR) MDCK cells was determined using a Pierce BCA Protein Assay Kit according to the manufacturer’s instructions. Total protein (30 μg) isolated from MDCK or MDR MDCK cells was used as a positive control. Similar preparations were made from B16, TC2, and 4T1 cells at low (2.5 × 105–5 × 105) and high (1.5 × 107–2 × 107) cell counts. The samples were separated on a 4%–15% Tris–HCl gradient polyacrylamide gel and electrotransferred to polyvinylidene fluoride membrane. The membrane was incubated in blocking buffer (5% nonfat dry milk in PBS with 0.1% Tween 20) for 1 h at room temperature. The membrane was then incubated with P-gp detection antibody C219 (Thermo Fisher) overnight at 4 °C, washed (0.1% Tween 20 in PBS), and incubated with alkaline phosphatase-conjugated secondary antibody (Promega) for 30 min at 25 °C. The membrane underwent a final wash before staining using Immune Star AP substrate (Bio-Rad) according to the manufacturer’s instructions (Fig. 4c ).
3
Western Blotting of Cell Lysates
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Western blotting was performed as described previously22 (link),76 (link), using cell lysates extracted by the TCA method. After being harvested and washed twice with 20% TCA, cells were roughly disrupted with zirconia beads by the Multi-beads shocker (Yasui Kikai). The protein precipitate recovered by centrifugation was suspended in SDS-PAGE sample buffer adjusted to pH 8.8 and then incubated at 95 °C for 10 min. After electrophoresis, the proteins were transferred onto a nylon membrane (Immobilon-P, Millipore) and incubated with primary antibodies in a blocking buffer (1 × PBS, 0.5% BSA) and then with alkaline phosphatase-conjugated secondary antibody (Promega). The color reaction was developed with NBT/BCIP solution (Nacalai Tesque).
4
Western Blot Analysis of Cell Lysates
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Cells grown on poly‐l ‐lysine‐coated dishes were lysed using RIPA buffer solution. The lysates were electrophoresed, transferred onto nitrocellulose membranes, and immunoblotted with antibodies to β‐actin (1:1000; mouse monoclonal; Sigma), CD44 (1:250; mouse monoclonal; Cell Signaling Technology), or VEGF (1:50; rabbit polyclonal; Abcam). Following incubation with alkaline phosphatase–conjugated secondary antibody (Promega), immunoreactions were developed using nitro blue tetrazolium and 5‐bromo‐4‐chloro‐3‐indolyl phosphate.
5
Western Blotting of GAD67 in Mouse Hippocampus
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Mice were anesthetized with ether before decapitation for western blotting. The hippocampi were dissected and stored at −80°C until protein analysis. Samples were then sonicated in ten volumes of SDS sample buffer and centrifuged at 15,000 rpm for 15 min at 4°C, followed by 3 min of incubation at 100°C. Aliquots containing 50 μg of protein were subjected to 10.0% acrylamide gel electrophoresis following standard procedures. Western blotting was performed on polyvinylidene fluoride (PVDF) membranes using a semi-dry blotting apparatus (HorizeBLOT 4M-R, ATTO, Japan). Immunodetection was performed using a GAD67 antibody (1/500 dilution; OriGene TA308881). After overnight incubation at 4°C with the primary antibody, the membranes were incubated in alkaline phosphatase-conjugated secondary antibody (1/1,000 to 1/2,000 dilution; Promega, Madison, WI, USA) at room temperature for 1 h. Immunoreactive bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich) and nitro blue tetrazolium (Sigma-Aldrich) in 100 mM Tris/HCl (pH 9.5) containing 100 mM NaCl and 5 mM MgCl2. The membranes were rinsed for 15 min between each step with Tris/HCl buffer (pH 7.4) containing 0.1% Tween 20.
6
Western Blotting and Immunostaining of Meiotic Proteins
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Anti-myc (9E10, sc-40, Santa Cruz), anti-tubulin (MCA77G, Bio-Rad/Serotec, Ltd), anti-Cdc5 (sc-33625, Santa Cruz), and histone H3 phospho-T11 (ab5168, Abcam) were used for western blotting. Rabbit anti-Rad5121 (link) and guinea pig anti-Rad5173 (link), rabbit anti-Hop174 (link) and rabbit anti-Rfa275 (link) were home-made and used for staining. Rabbit anti-Ndt80 was a gift by M. Lichten. The secondary antibodies for immuno-staining were Alexa Fluor 488 (Goat, A-21206, Thermo Fisher Scientific) and 594 (Goat, A-211012, Thermo Fisher Scientific) IgG used at a 1/2000 dilution. For western blotting, alkaline phosphatase-conjugated secondary antibodies (Promega) were used.
7
Murine Endothelial Cell Protein Expression
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Murine cerebrovascular endothelial cells underwent Western blotting as previously described by Yin et al.9 (link). Briefly, the cells were lysed via lysis buffer to isolate the total protein. Equal protein amounts were subjected to SDS-PAGE followed by transfer to polyvinyldifluoride (PVDF) membranes. PVDF membranes were then blocked in a blocking solution (5% non-fat milk in Tris-buffered saline) followed by incubation with the following primary antibodies for two hours at room temperature: anti-PPARγ (1:200; catalog # sc-7273, Santa Cruz Biotechnology), anti-CD36 (1:200; catalog # sc-9154, Santa Cruz Biotechnology), and β-actin (1:500; catalog # sc-47778, Santa Cruz Biotechnology). The membranes were then incubated for one hour at room temperature with the appropriate alkaline phosphatase-conjugated secondary antibodies (Promega). The ProtoBlot AP System (Promega) was applied to assess the color reaction according to the kit’s instructions.
8
Cdc31 Protein Extraction and Analysis
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Pelleted cells were washed in PBS and frozen in liquid nitrogen. Thawed pellets were resuspended in 1 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1%NP-40, 1 mM DTT, 10% glycerol and 1 mg/ml each pepstatin A, aprotinin and leupeptin) and ~100 μl of glass beads were added prior to bead beating for 1 min x 5 with 2 min on ice between beatings. Samples were spun at 5000 rpm for 2 min and the supernatant was transferred to a new tube. Protein concentration was determined using a NanoDrop Spectrophotometer (Thermo), and equivalent amounts of lysate were analyzed by SDS-PAGE followed by western blotting. The following primary antibody dilutions were used: 1:1000 anti-Cdc31 [27 (link)] and 1:5000 anti-Pgk1 (Invitrogen). Alkaline phosphatase-conjugated secondary antibodies were used at 1:10000 (Promega). Western blot band intensity was analyzed with ImageJ Gel quantification tool.
9
Protein Extraction and Analysis from NDC1 Yeast Cells
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Lysates from NDC1-GFP or ndc1-L562S-GFP cells were prepared from mid-log phase cultures. Pelleted cells were washed in PBS and frozen in liquid nitrogen. Thawed pellets were resuspended in 1 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1%NP-40, 1 mM DTT, 10% glycerol, and 1 mg/ml each pepstatin A, aprotinin, and leupeptin), and ∼100 µl of glass beads were added before bead beating for 1 min × 5 with 2 min on ice between beatings. Samples were spun at 5,000 rpm for 2 min and the supernatant was transferred to a new tube. Protein concentration was determined using a spectrophotometer (NanoDrop; Thermo Fisher Scientific), and equivalent amounts of lysate were analyzed by SDS-PAGE followed by Western blotting. Whole-cell extracts were prepared by bead beating into SDS sample buffer to determine expression levels of baits. The following primary antibody dilutions were used: 1:1,000 anti-GFP (Roche); 1:1,000 anti-Pgk1 (Invitrogen); and 1:1,000 anti-LexA (EMD Millipore). Alkaline phosphatase–conjugated secondary antibodies were used at 1:10,000 (Promega) and horseradish peroxidase–conjugated secondaries were used at 1:1,000 (Cell Signaling Technology).
10
Immunoprecipitation of Mouse Cortical Neurons
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Mouse cortical neurons (14 DIV) were lysed in lysis buffer T. The lysates were precleared with protein A-Sepharose (GE Healthcare) for 1 h at 4 °C. Precleared lysates (100–500 μg of protein) were incubated with 5 μg of the indicated antibody for 1 h at 4 °C, and then, the immunocomplexes were adsorbed to protein A-Sepharose for 1 h at 4 °C. After washing extensively with lysis buffer T, the immunoprecipitates were resolved by SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The blots were probed with primary antibodies as indicated and visualized with alkaline phosphatase-conjugated secondary antibodies (Promega). Images have been cropped for presentation (Fig. 4 and Supplementary Fig. 3 ). Full-size images are presented in Supplementary Figs 9 and 10 .
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