Retinas were rapidly dissected through a slit in the cornea using the Winkler technique. 26 The tissue was sonicated in 100 mM Tris/HCl, pH 7.5, containing complete protease inhibitor cocktail tablets (Roche Diagnostics, Rotkreuz, Switzerland). Retinal extracts were cleared by centrifugation and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) and then electrotransfered to nitrocellulose Hybond enhanced chemiluminescent membranes (GE Healthcare Europe GmbH, Otelfingen, Switzerland) in a Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% low fat milk. PAX6 was detected with corresponding polyclonal antibodies (1:5000) (Chemicon, Temecula, CA) and alkaline phosphatase-conjugated secondary antibodies (1:15,000) (Sigma-Aldrich, St. Louis, MO). PAX6 was normalized to actin as a protein loading control. Actin was detected with mouse monoclonal antibodies to actin (1:5000) (Sigma, Buchs, Switzerland) and alkaline phosphatase-conjugated secondary antibodies (1:15,000) (Sigma-Aldrich, St. Louis, MO). Alkaline phosphatase activity was visualized with the Bio-Rad detection kit (Bio-Rad Laboratories, Hercules, CA) and a VersaDoc Imaging System (Bio-Rad Laboratories, Hercules, CA).
Alkaline phosphatase conjugated secondary antibody
Manufactured by Merck Group
Sourced in United States, China
Alkaline phosphatase-conjugated secondary antibodies are laboratory reagents used in various immunoassay techniques. They consist of secondary antibodies that are chemically linked to the enzyme alkaline phosphatase. This conjugate can be used to detect and quantify target proteins or molecules in a sample by binding to a primary antibody that is specific to the target.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Immun star ap detection system, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
β actin, by Merck Group (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Dnmt3b, by Abcam (1 mentions)
Nlrp3, by LSBio (1 mentions)
Caspase 1, by GeneTex (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Gel doc imaging system, by Bio-Rad (1 mentions)
1 step nbt bcip, by Thermo Fisher Scientific (1 mentions)
Ripa lyses buffer, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Thermo Fisher Scientific (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
44 protocols using alkaline phosphatase conjugated secondary antibody
1
Quantifying Retinal PAX6 Expression
2
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots performed with 10, 12, or 15% SDS-polyacrylamide gels were incubated with primary antibodies for IL-1ß (R&D Systems, Minneapolis, MN), NLRP3 (LSBio, Seattle, WA), Ogg1 (Novus Biologicals, Littleton, CO), Caspase 1 (Genetex, Irvine, CA), and DNMT3b (Abcam, Cambridge, MA). Western blots were visualized using alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich).
3
Western Blot Analysis of Anti-HIV-1 and Anti-HTLV-1 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anti-HIV-1 antibody was obtained from the NIH AIDS Research and Reference Reagent Program. The anti-beta tubulin antibody was obtained from GeneTex (GeneTex, Irvine, CA, USA; GTX101279). The HTLV-1 p19 ELISA kit and anti-p19 antibody were from Zeptometrix (Buffalo, NY, USA). The experimental protocol for western blot was previously described [33 (link),34 (link)]. Briefly, cells were harvested, washed, and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). The lysates were resolved by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies overnight at 4°C, and then incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma). Signals were visualized using chemiluminescence following the manufacturer’s protocol (Chemicon, Billerica, MA, USA). Band intensities were quantified using ImageJ software.
4
C1q Binding Assay for Mesothelioma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mesothelioma cells were grown to confluence in 96-well tissue culture plates and incubated directly with increasing concentrations of purified human C1q or preincubated with increasing concentrations of HA, for 1 h at room temperature. Bound C1q was revealed by ELISA using mAb anti-human C1q (10 µl/ml) and alkaline-phosphatase-conjugated secondary antibodies (Sigma-Aldrich). The color, developed using pNPP (Sigma-Aldrich; 1 mg/ml) as a substrate, was read at 405 nm using a Titertek Multiskan ELISA reader (Flow Labs, Milano, Italy).
5
Validating EMT Changes in MCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the changes in EMT of MCs, the expression of the epithelial marker, E-cadherin (anti-E-cadherin; BD Bioscience, Bedford, MA), were identified [21 (link)]. The blots were incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich, San Luis, MO) to each primary antibody and detected using enhanced chemiluminescence (Chemicon International, Billerica, MA).
6
Western Blot Analysis of Hfq and Crc
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of proteins were separated on 12% SDS-polyacrylamide gels, and then electro-blotted onto a nitrocellulose membrane. The blots were blocked with 5% dry milk in TBS buffer, and probed with rabbit anti-Hfq (Pineda) and rabbit anti-Crc (Pineda) antibodies, respectively. Immuno-detection of ribosomal protein S1 served as a loading control. The antibody-antigen complexes were visualized with alkaline-phosphatase conjugated secondary antibodies (Sigma) using the chromogenic substrates nitro blue tetrazolium chloride (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP).
7
Quantitative Analysis of mTOR Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were isolated, suspended in 1 × RIPA lyses buffer (Sigma) supplemented with Complete Protease Inhibitor tablets (1 per 50 ml) (Roche) and PhosSTOP phosphatase inhibitor tablets (1 per 10 ml), sonicated and then centrifuged at 14 000 g for 30 min at 4 °C. Seventy microgram of total protein was separated on 4–20% SDS-polyacrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 4% bovine serum albumin (Sigma) in 1.0% Tween (Sigma) in PBS and probed with primary antibodies against mTOR pSer2448 (1:10 000; Abcam) and glyceraldehyde 3-phosphate dehydrogenase (1:10 000; Ambion, Life Technologies) overnight at 4 °C, then with alkaline phosphatase-conjugated secondary antibodies (Sigma) for 2 h at room temperature. The membrane was visualized using 1-step NBT/BCIP (Thermo Fisher Scientific). Imaging was done using a Bio-Rad Gel Doc Imaging System; the membrane was cut during imaging. Semi-quantitative analysis was done with NIH ImageJ (http://imagej.nih.gov/ij/ ), which was used to compare the density (intensity) of bands.
8
Capturing and Quantifying Dengue Virus Particles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Virus particles were captured using mouse anti-DENV MAbs 4G2 and 2H2 in carbonate buffer. MAbs (see Table S1 in the supplemental material) were diluted to a concentration of 20 ng/µl and added to captured virus for 1 h at 37°C. After incubation with alkaline phosphatase-conjugated secondary antibodies (Sigma), p-nitrophenylphosphate substrate (Sigma) was added and absorbance at 405 nm was measured (Bio-Rad). Background signal (optical density at 405 nm [OD405] with no primary antibody) was subtracted from each virus sample, and absorbance was normalized to binding of an antiflavivirus human polyclonal serum sample.
9
Cerebellar Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell lysates from vermis of mouse cerebellums were extracted using RIPA buffer [50 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM EDTA, 20 mM β-gylcerophosphate, 0.1 mM Na3VO4, 1 mM NaF, 0.5 mM DTT and protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)] containing 0.5% NP-40 plus rigorous sonication. The lysates were analyzed by using SDS-PAGE, followed by transfer to polyvinylidene fluoride (PVDF, Millipore) membranes and blotting with primary antibodies. Corresponding alkaline-phosphatase-conjugated secondary antibodies (Sigma-Aldrich) were added, and the blots were developed by chemiluminescence following the manufacturer's protocol (PerkinElmer).
10
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted with 8 M urea, 0.1 M Tris-HCl, pH 7.0, 1% SDS, fractionated by SDS-PAGE (12% acrylamide gel) and transferred to a PVDF membrane (Immobilon-P, Millipore). Blots were developed using alkaline phosphatase-conjugated secondary antibodies (Sigma) and the Immun-Star AP detection system (Bio-Rad). The following antibodies were used for detection: murine monoclonal anti-lamin Dm0 (1:2000; ADL6749 (link)), rabbit polyclonal anti-lamin C25 (1:10000), murine monoclonal anti-beta Actin (1:3000; ab8224, Abcam).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!