SDS-PAGE and western blotting of virus-infected BHK-21 cell lysate was carried out as described earlier (Ranjitha et al. 2020 (link)). Briefly, the protein-blotted PVDF membrane (Merck, USA, #GE10600089) was probed using rabbit monospecific serum raised against t3A, 3D-specific mAb (6B8D11) (Kushwaha et al. 2021 (link)), 3B-specific mAb (10H9D8), rabbit serum against serotype O capsid antigen or GAPDH (Santa Cruz, USA, #SC-59540) (dilution 1:500 for all), followed by species-specific HRP-conjugated secondary antibody (1:1000) (Dako, Denmark, #P0448 [anti-rabbit] and #P0260 [anti-mouse]), and membrane visualization using Clarity Western ECL substrate (Bio-Rad, USA, #1705060) in a chemiluminescence imager (Uvitec, UK).
Sc 59540
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-59540 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for use in scientific research and experiments. The core function of this product is to provide a specific tool or instrument for conducting various types of biological or chemical analyses.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab176328, by Abcam (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
A0208, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Dingguo (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Pierce micro bca assay, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Pp k9218 00, by R&D Systems (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
C510003, by Sangon (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Bca protein assay kit, by Applygen (1 mentions)
6 protocols using sc 59540
1
SDS-PAGE and Western Blotting of Virus-infected Cells
2
Tissue Protein Extraction and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the mice were sacrificed, tissues were immediately collected, snap-frozen in liquid nitrogen, and stored at –80°C for downstream analyses. Muscles were lysed in 200–350 μL ice-cold 1% Triton X100 in PBS containing proteinase inhibitors (Roche Diagnostic), homogenized using TissueLyser (QIAGEN), and 25 μg of protein lysate was processed as described previously (13 (link)). Primary antibodies — mouse monoclonal anti-FGF1 (AF4686, R&D Systems), mouse monoclonal anti-GAPDH (sc-59540, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti–HO-1 (ADI-SPA-894, Enzo Life Sciences), rabbit polyclonal anti-AMPKA (2603S, Cell Signalling Technology), and rabbit monoclonal anti–PGC1-β (ab176328, Abcam) — and secondary antibodies (conjugated with HRP) include anti–mouse Ig (554002, BD Biosciences) for the detection of GAPDH or FGF1, as well as anti–rabbit IgG (7074, Cell Signaling Technology) for the detection of HO-1, AMPKA, PGC1-β, were used.
3
Quantifying EphA2 and Pho-EphA2 in Fallopian Tube
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein content extracted from the fresh Fallopian tube samples was resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 10% bovine serum albumin (BSA) (DingguoChangsheng Biotechnology Co., Ltd., Beijing, China) at room temperature, followed by incubation at 4 °C overnight with the primary antibodies, rabbit anti-human polyclonal antibodies against EphA2 and Pho-EphA2 (1:1000; 6997S and 6347S; Cell signaling technology Co., Ltd., Boston, USA) and mouse anti-human monoclonal antibody against glyceraldehyde phosphate dehydrogenase (GAPDH) (1:1000; sc-59,540; Santa Cruz, California, USA). After washing, the membranes were then incubated for 1 h with the appropriate HRP-conjugated secondary antibodies, goat anti-rabbit IgG (1:800; A0208; Beyotime Biotechnology Co., Ltd., Shanghai, China) or rabbit anti-mouse IgG (1:1000; D031402; Dako Cytomatin, Glostrup, Denmark). Protein bands were identified using ECL (Pierce, Rockford, USA). The bands were semi-quantified using Bio-Rad Quantity One software. EphA2 was normalized to GADPH, and Pho-EphA2 was normalized to EphA2.
4
Moesin Phosphorylation and Cell Signaling Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatments, cells were collected on ice with lysis buffer containing 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% IGEPAL, protease inhibitor cocktail (Sigma-Aldrich, USA), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich, USA). The concentration of total proteins was quantified by Pierce Micro BCA Assay (Thermo Fisher Scientific). Samples, containing 25 μg of protein, were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane (Immobilon-P, Millipore). Antibodies against the following proteins were used: Moesin (sc-610402), Thr558-p-Moesin (sc-12895), AR (sc-816), estrogen receptor (ER) (sc-8005), and GAPDH (sc-59540), all purchased from Santa Cruz Biotechnology. Primary and secondary antibodies were incubated with standard technique. Immunodetection was accomplished with a quantitative digital imaging system (Quantity One; BioRad, USA). Densitometric analysis of the proteins bands was performed using the NIH ImageJ 1.49p software.
5
Western Blot Analysis of HNF4A-P1 Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were quantified using Bradford assay (Bio-Rad), resolved on a 10% SDS-PAGE gel and subsequently transferred onto a polyvinylidene difluoride membrane. The following antibodies were used: mouse-anti HNF4A-P1 isoforms (1:1,500, 3 hours, room temperature; R&D Systems PP-K9218–00, RRID: AB_1964277), mouse anti-GAPDH (1:5,000, 3 hours, room temperature; Santa Cruz Biotechnology sc-59540, RRID: AB_631587), anti-mouse IgG, horseradish peroxidase–linked Antibody 7076P2 (1:10,000, 1 hour; room temperature; Cell Signaling Technology 7076p2, RRID: AB_330924). Amersham ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences), an enhanced luminol-based detection system, was used for luminescent signal generation.
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the biopsy adipose samples and cultured cells using a commercial protein extraction kit [250 mM Tris (pH 7.5), 40 mM NaCl, 10 mM EDTA, 5 mM NP-40, protease and phosphatase inhibitors; C510003, Sangon Biotech Co. Ltd., Shanghai, China] according to the manufacturer's instructions. The protein concentration was determined using the BCA Protein Assay Kit (P1511, Applygen Technologies). Thirty micrograms of protein was separated using SDS-PAGE with known molecular weight markers (Sangon Biotech Co. Ltd.). Subsequently, the protein was transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated with primary antibodies against GAPDH (internal control; sc-59540, Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:5,000), HSPB7 (cat. no. Ab150390, Abcam, Cambridge, MA; 1:1,000), and NFE2L2 (cat. no. Ab31163, Abcam; 1:1,000) at 4°C overnight. Subsequently, the PVDF membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Boster, Wuhan, China). Last, the immunoassay was performed using an enhanced chemiluminescent reagent (Pierce Biotechnology Inc., Chicago, IL). Densitometry analysis was performed with Image-Pro Plus 6.0 (Media Cybernetics Inc., Warrendale, PA) and target protein expression was normalized to GAPDH expression.
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!