Cells and tissues were lysed in RIPA lysis buffer (Beyotiom Biotech, China), and protein concentration was determined by BCA protein assay kit (Beyotiom Biotech, China). Total proteins were separated for each sample using 10% SDS-PAGE and then transferred to PVDF membrane (Millipore, Burlington, USA). The membrane was blocked in 5% skim milk for 1 h and incubated with the primary antibody at 4 ℃ overnight (Supplementary Table 1). After washing with Tris-buffered saline with 0.1% Tween 20 (TBST) three times, membranes were incubated with secondary antibodies for 1 h at room temperature. The target protein bands were visualized using Super Enhanced chemiluminescence detection reagent (Applygen Technologies Inc., Beijing, China).
Super enhanced chemiluminescence detection reagent
Manufactured by Applygen
Sourced in China
The Super-enhanced chemiluminescence detection reagent is a laboratory product that enhances the detection of chemiluminescent signals. It is designed to improve the sensitivity and performance of chemiluminescence-based assays and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Runx1, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg antibody, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Procaspase 9, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Histon h3, by Cell Signaling Technology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Ab7280, by Abcam (1 mentions)
Sc 40, by Santa Cruz Biotechnology (1 mentions)
9 protocols using super enhanced chemiluminescence detection reagent
1
Western Blot Protein Analysis
2
RUNX1 Protein Expression in OSCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to a previous study, western blotting was performed to identify protein expression in OSCC cells [30 ]. First, the cells were lysed to isolate total proteins using RIPA buffer (Thermo, USA). After detecting the protein concentrations using a BCAkit (Thermo, USA), 20 µg/lane protein was separated by 10% SDS-PAGE at 100 V. Then, the separated proteins were transferred onto PVDF membranes for 2 h at 22–25°C using 5% skimmed milk. Next, the membranes were incubated with primary antibodies, including RUNX1 (Abcam, USA) and GAPDH (Abcam, USA) at 4°C overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Abcam, USA) for 3 h. Finally, the membranes were incubated with SuperEnhanced Chemiluminescence Detection Reagent (Applygen, China) for 10 min, covered with plastic wrap, and exposed to X-ray film.
3
Protein Expression Analysis by SDS-PAGE and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE and Western blotting were performed according to standard protocols. Briefly, the protein lysates were separated on SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The blots were blocked and probed with primary antibodies against PLK1 (Abcam, Cambridge, UK), V5 (Invitrogen), pro-caspase-3, full-length PARP (FL-PARP) (Proteintech, Wuhan, China), p-Histone-H3(p-HH3)(Ser10)(Immunoway Biotechnology, TX, USA), pro-caspase-8 (Atlas Antibody, Stockholm, Sweden), cyclin B1, pro-caspase-9, Histon-H3, p-ERK (Thr202/Tyr204), ERK1/2, p-AKT (Ser473), AKT, Stat3, and p-Stat3 (Tyr705) (Cell Signaling Technology, Danvers, MA, USA). β-actin (Amart, Shanghai, China) was used as a loading control. The signals were visualized with a super-enhanced chemiluminescence detection reagent (Applygen).
4
Immunoblotting of MTAP, E-cadherin, GSK3β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was conducted with primary antibodies against MTAP (Abcam, ab126770; 1:1,000), E-cadherin (Cell Signaling Technology, #3195; 1:1,000), p-GSK3β (Cell Signaling Technology, #9323; 1:1,000), or GSK3β (Cell Signaling Technology, #12456; 1:1,000). β-actin (Sigma, A19781; 1:5,000) was used as a loading control. The signals were visualized using the super-enhanced chemiluminescence detection reagent (Applygen Technologies, Inc., Beijing, China).
5
Western Blot Analysis of Notch Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with ice-cold PBS and lysed in RIPA buffer [50 mmol/l Tris (pH 7.5), 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)] containing phenylmethylsulfonyl fluoride (PMSF; 1 mmol/l) and protease inhibitors (2 g/ml; Protease inhibitor cocktail set III, Calbiochem, Billerica, MA, USA) on ice for 30 min. The lysates were clarified by centrifugation at 13,000 × g for 30 min at 4°C. The total protein concentration was estimated using a Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel, transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), blocked and probed with antibodies against β-catenin (1:1,000; sc-65480; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), c-myc (1:1,000; sc-40; Santa Cruz Biotechnology, Inc.), Jagged (1:1,000; sc-390177; Santa Cruz Biotechnology, Inc.), Notch1 (1:1,000; sc-373891; Santa Cruz Biotechnology, Inc.), Notch2 (1:1,000; sc-5545; Santa Cruz Biotechnology, Inc.), DLL4 (1:1,000; ab7280; Abcam, Cambridge, MA, USA) and Pra-1 (1:1,000; ab76413; Abcam). Upon washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by super enhanced chemiluminescence detection reagent (Applygen, Beijing, China).
6
Western Blotting for PLAU Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was used to detect the expression of the PLAU protein according to previous study [19 (link)]. Total protein from transfected cells was isolated using 200 µL of RIPA lysis buffer (Beyotime, China). Then, 20 µg of protein was added to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking the membranes with 5% nonfat milk in TBST, the membranes were incubated with PLAU antibody (Cat#: ab169754, Abcam, USA) or GAPDH antibody (Cat#: ab9485, Abcam, USA) at 4°C for 12 h. Next, the HRP anti-rabbit IgG antibody (Cat#: ab270144, Abcam, USA) was incubated at 22°C for 3 h. After washing the membranes, SuperEnhanced chemiluminescence detection reagent (Applygen, China) was added to the membranes for 15 min incubation, and the damp blot was covered with plastic wrap. The blot was exposed to an X-ray film.
7
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tumor tissues are lysed on ice for 30 minutes with RIPA lysis buffer containing protease inhibitors (PMSF) and phosphatase inhibitors. Equal amounts of the total protein (20 μg for cells, 50 μg for tissues) were separated by 8% - 15% SDS-PAGE gel. After electrophoresis, the proteins on the SDS-PAGE gel were transferred to the polyvinylidene fluoride membrane (PVDF). Then, the membrane was blocked for 1 hour in a TBST solution containing 5% skimmed milk powder or Bovine Serum Albumin (BSA) at room temperature. Furthermore, the membrane was incubated overnight with the different primary antibodies at 4°C. Next, the membrane will be further incubated for 1 hour with horseradish peroxidase (HRP) conjugated secondary antibody at room temperature. Finally, the protein bands on the membrane were visualized by Super Enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and detected by Chemi Dox XRS chemiluminescence imaging system (Bio-Rad, California, USA). The protein bands were quantified by Image J (NIH, USA) and the relative expression of the target protein was calculated by using GAPDH as an internal control. All data comes from three independent experiments.
8
Whole-Kidney Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-kidney homogenates and cells were prepared with RIPA buffer containing 1% NP-40, 0.1% SDS, 100 μg/mL PMSF, and 1% Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (78442; Thermo Fisher Scientific) in PBS on ice. The supernatants were collected after centrifugation at 13,000g at 4°C for 15 minutes, and concentration was determined with BCA protein assay (K813-5000-1; BioVision). Gel electrophoresis was performed on reduced, denatured samples. After blotting onto PVDF microporous membranes (IPVH00010; MilliporeSigma) and blocking with 5% milk, membranes were incubated with primary antibodies and HRP-conjugated secondary antibodies. The protein bands were visualized by SuperEnhanced chemiluminescence detection reagents (P1010; Applygen Technologies Inc.) and Kodak x-ray film. The primary and secondary antibodies used are listed in Supplemental Table 3 . Relative protein levels of Western blots were quantified with densitometries, analyzed by ImageJ software (NIH), and reported after normalizing to the loading controls.
9
Quantifying mRNA m6A Methylation via Dot Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A dot blot assay used to measure the level of mRNA m6A methylation was performed as follows. Total RNA was isolated. The isolated RNA was denatured at 95°C for 3 min, followed by immediate chilling on ice. The denatured RNA (100 ng) was spotted on a Nitrocellulose membrane (GE Healthcare, RPN203B, United States). Then, the membranes were ultraviolet (UV) crosslinked with a StratageneStratalinker 2,400 UV Crosslinker and were blocked in blocking buffer (5% non-fat milk in PBST) for 1 h. N6-Methyladenosine (m6A) recombinant rabbit monoclonal antibody (Invitrogen, MA5-33030, United States) was incubated with the membrane overnight at 4°C with a dilution of 1:1,000. After washing three times with 1 × PBST, a corresponding horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, 7074S, United States) was diluted 1:5,000 and incubated with the membranes for 1 h at room temperature. After extensive washing, the membrane was visualized by Super-enhanced chemiluminescence detection reagents (Applygen Technologies, P1030, China). To determine whether each dot possess an equal amount of RNA, the RNAs with same amount were spotted on the membrane and stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) as a control.
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!