Proteins were extracted from the cortex, hippocampus and cerebellum as described above (see Sample preparation for LC-MS/MS analysis section). 20 μg of proteins from each tissue lysate were resolved by 4–20% Mini-PROTEAN TGX precast gels (BioRad) and transferred to a PVDF membrane (BioRad). The membrane was first blocked in the blocking buffer (5% nonfat dry milk, 0.1% Tween-20; in PBS) for 1 h at room temperature and then incubated with anti-Aβ antibody (1:500; 4G8; Signet, USA), anti-HSP70 antibody (1:200; Santa Cruz Biotechnology, USA), anti-mTOR antibody (1:250; Santa Cruz Biotechnology, USA), and anti-Cdk-5 antibody (1:200; Santa Cruz Biotechnology, USA) in blocking buffer for overnight at 4°C. After washing, the membrane was incubated with an HRP-conjugated goat anti-mouse secondary antibody (1:2000; Santa Cruz Biotechnology, USA) for 1 h at room temperature and detected by chemiluminescence (Bio-Rad). Equal protein loading was confirmed by stripping the blot and re-probing with a β-actin antibody (1:1000; Santa Cruz Biotechnology, USA). Western blots were quantified by densitometry using the ImageJ software.
Hrp conjugated goat anti mouse secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The HRP-conjugated goat anti-mouse secondary antibody is a laboratory reagent used to detect and quantify mouse primary antibodies in various immunoassays. It consists of a goat-derived secondary antibody that is conjugated to the enzyme horseradish peroxidase (HRP). This enzyme can catalyze a colorimetric or chemiluminescent reaction, allowing the visualization and quantification of the targeted mouse primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (3 mentions)
Nitrocellulose membrane, by Cytiva (3 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (3 mentions)
β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Bio-Rad (2 mentions)
Gapdh 0411, by Santa Cruz Biotechnology (2 mentions)
Pgr 312 l ce, by Leica (2 mentions)
Ecl select western blot detection kit, by GE Healthcare (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Anti hsp70 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti mtor antibody, by Santa Cruz Biotechnology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Western blot detection reagent, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hrp conjugated mouse anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh antibody, by Abmart (1 mentions)
26 protocols using hrp conjugated goat anti mouse secondary antibody
1
Protein Expression Analysis in Alzheimer's
2
Immunoblotting of AQP5 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates from 3AO cells and tissues were prepared in ice-cold RIPA buffer (Beyotime) supplied with protease inhibitor cocktail (Roche) and 1 mM PMSF (Calbiochem). After centrifuge at 13,000 × g for 10 min, the supernatant of lysates was denatured, loaded into the wells of the 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins were separated by electrophoresis, transferred to nitrocellulose membranes (Millipore), blocked with 5% nonfast milk in Tris buffered saline (TBS) buffer with 0.05% Tween-20 for 1 hour at room temperature, and then probed with mouse anti-AQP5 antibody (1:1300, Abcam, UK) at 4°C overnight. After incubation for 2 hours with horseradish-peroxidase (HRP)-conjugated rabbit anti-mouse secondary antibody (1:5000, Santa Cruz Biotechnology, USA), bands were visualized by ECL plus western blot detection reagent (GE, USA). The house-keeping gene GAPDH was used as a loading control and detected by using mouse anti-GAPDH antibody (1:4000, abmart, China) plus HRP-conjugated goat anti-mouse secondary antibody (1:5000, Santa Cruz Biotechnology, USA).
3
Quantifying Renal Macrophages via IHC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophage number was examined in this study given the known association between oxidative stress and inflammation (Salzano et al. 2014 ; Sutherland et al. 2014 ), as well as macrophage number being a strong correlate of both renal interstitial and glomerular disease progression (Duffield 2010 ). The number of CD68+ macrophages was assessed using immunohistochemistry. Dewaxed sections underwent heat‐induced antigen retrieval in Tris‐Ethylenediaminetetraacetic acid (EDTA) buffer (10 mmol/L Tris Base, 1 mmol/L EDTA, 0.05% Tween‐20, pH 9.0), were blocked with H2O2 followed by goat serum, and then incubated overnight at room temperature with mouse anti‐CD68 (Ab31630, Abcam, Toronto, ON, Canada) at 1:200 dilution. Sections were then incubated with a horseradish peroxidase (HRP)‐conjugated goat anti‐mouse secondary antibody (Santa‐Cruz, Dallas, TX) at 1:400 for 90 min. 3,3'‐diaminobenzidine (DAB) was used to visualize antibody binding, and sections were counterstained with hematoxylin. Sections with the primary antibody excluded served as negative controls. Systematic random sampling at a step length of 2 mm was performed, with all positively stained cells (glomerular and interstitial) recorded at each field of view. Any cells within vessel lumens were excluded. The average number of CD68+ cells per field of view was calculated for each kidney.
4
Western Blot Analysis of Tumor Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from the tumor tissues and the protein concentration was determined using the Bradford method (15 (link)). A total of 50 μg protein was loaded per lane. Following electrophoresis, all proteins were transferred to a cellulose acetate membrane (Koch Membrane Systems, Inc., Wilmington, MA, USA) and the membrane was blocked with 5% non-fat milk in PBS overnight at 4°C. The following day, the blot was incubated with a primary monoclonal mouse anti-human antibody (1:1,000; Santa Cruz Biotechnology, Inc.) for 2 h at 37°C and washed three times with PBS. Next, the blot was incubated with a HRP-conjugated goat anti-mouse secondary antibody (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C and X-ray films were used to observe the bands of the western blot. The proteins were analysed using Scion Image version 4.0.3.2 (Scion Corporation, Frederick, MD, USA). The protein content was observed relative to that of the GAPDH control band.
5
IHC Analysis of p22 phox and GFAP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For IHC analysis, the free-floating method was used for the selected tissue in each treatment group. After washing the tissue 3× for 10 min each in 0.01 M PBS, the tissue was incubated at 80 °C for 60 min in a beaker containing 0.01 M sodium citrate, followed by blocking in 10% normal donkey serum (Millipore Sigma, Burlington, MA, USA). The tissue sample was then reacted with primary antibodies anti-p22 phox (Santa Cruz Biotechnology, dilution: 1:500) and anti-Glial fibrillary acidic protein (GFAP) (Abcam, dilution: 1:500) for 12 h at 4 °C. The following day, the tissue was washed 3× for 5 min each in 0.01 M PBS and then reacted with HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, dilution: 1:5000) at room temperature for 2 h. Finally, the tissue was incubated at room temperature using a Vectastain-Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) solution, and the results were visualized using the DAB Peroxidase Substrate Kit (Vector Laboratories). Each tissue slice was mounted on a slide using a mounting medium (Vector Laboratories) and inspected using a light microscope (Leica Microsystems).
6
Western Blot Analysis of VEGFA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cultured cells using RIPA buffer (Beyotime, Shanghai, People’s Republic of China) and the protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein (30 µg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 5% nonfat milk, the blots were probed with mouse antibodies against VEGFA and GAPDH (1:1000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), followed by incubation with an HRP-conjugated goat-anti-mouse secondary antibody (1:5000; Santa Cruz Biotechnology Inc.) for 2 h at room temperature. The protein bands were detected with enhanced chemiluminescence reagents (Pierce) and were quantified using the Scion Image software and normalized to GAPDH levels.
7
Western Blot Analysis of Rae-1β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Different amounts of Rae-1β recombinant protein were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to nitrocellulose membranes using the iBlot gel transfer device (Invitrogen, Grand Island, NY). The membranes were blotted with anit–Rae-1 primary antibody and HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Dallas, TX) to detect the protein of interest.
8
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell extracts of BEAS-2B cells were obtained with Chemicon Total Protein Extraction Kit (Merck Millipore, USA) whereas protein samples of cytoplasmic and nuclear fractions were extracted with NucBuster Protein Extraction Kit (Merck Millipore, USA) according to the manufacturer's instructions. Protein samples of 20 μg were electrophoresed on 8–12% SDS-polyacrylamide gels and transferred to 0.2 μm polyvinylidene fluoride membranes using a wet transfer system at 0.35 mA for 1.5 h (Bio-Rad Laboratories, USA). Membranes were blocked with 5% bovine serum albumin in Tris-buffered saline-tween 20 (TBS-T) for 1 h prior to overnight incubation at 4°C with respective antibodies (Table 2 ) in 5% BSA TBS-T. Following washing with TBS-T, membranes were hybridized with horse radish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5000; Santa Cruz Biotechnology, USA) or HRP-conjugated goat anti-mouse secondary antibody (1:5000; Santa Cruz Biotechnology, USA) for 1 h at room temperature. Membranes were then incubated with chemiluminescence reagent (Thermo Scientific, USA) for 1 min and imaged in a gel documentation system (Vilber Lourmat, Germany). Membranes were stripped and reprobed as required. Band intensities were quantified by ImageJ Image Processing Software (NIH, USA) and normalized by comparison to β-actin or Lamin A/C.
9
Protein Expression Analysis of PnAg Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIH-3T3 and HaCaT cells were treated with 1.5 µM and 20 µM PnAg separately for 48h. The control groups were treated with only solvent (0.1% DMSO in PBS). Subsequently, the cells were harvested and the proteins were extracted by using RIPA buffer (Beyotime, Jiangsu, China). Proteins were separated by electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked and incubated with primary antibodies; GAPDH was used as an internal control. After incubation with HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz, CA, USA), the proteins were visualized by an enhanced chemiluminescence kit (Amersham Corp, Buckinghamshire, United Kingdom) and exposed to chemiluminescent film.
10
Dot Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nitrocellulose membranes were divided into grids using a pencil. Each grid was loaded with a sample of 3 μg total protein lysate from the soluble fraction in 10 μL. Membranes were dried and blocked using a blocking solution comprising 5% skim dried milk in TBS-T for 1 h at room temperature. Membranes then were incubated with monoclonal antibody TOC-1, diluted 1:5000 in blocking solution overnight at 4 °C. The membranes were washed thrice in TBS-T and incubated with HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) diluted 1:10,000 in blocking solution for 1 h at room temperature. Dots were visualized by incubating the membranes with ECL substrate (SuperSignal, West Pico PLUS, ThermoFisher) for 5 min and imaged using an Azure 300 Chemiluminescent Western Blot Imaging System (Azure biosystems).
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!