Protein samples were isolated from colorectal cancer tissues using PARIS kit (Ambion). Total protein concentration was determined using Bradford protein reagent (Bio-Rad). Soluble proteins were loaded on precast TGX gels and were analyzed by immunoblotting with anti-TIGAR (dilution 1:1,000; Santa Cruz Biotechnology). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare). Membrane was developed using C-Digit Blot Scanner (LI-COR, Hamburg, Germany). CRC cell line whole cell lysates were prepared as described (22 (link)). Soluble proteins were analyzed by immunoblotting with anti-TIGAR (Santa Cruz Biotechnology) and anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare). Membranes were developed using C-Digit Blot Scanner (LI-COR).
Chemiluminescence
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Italy, France, Sweden
Chemiluminescence is a lab equipment product that utilizes the emission of light during a chemical reaction to detect and quantify specific analytes. The core function is to provide sensitive and accurate measurements by converting chemical energy into light energy, which can be detected and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (18 mentions)
Ripa buffer, by Thermo Fisher Scientific (9 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
2 mercaptoethanol, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (5 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti rabbit and anti mouse peroxidase conjugated antibodies, by Jackson ImmunoResearch (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Uvi 1d software, by Uvitec (4 mentions)
Hrp linked secondary antibody, by Bio-Rad (4 mentions)
Alliance mini hd6 system, by Uvitec (4 mentions)
Nitrocellulose membrane, by Merck Group (4 mentions)
Nitrocellulose membrane, by Cytiva (4 mentions)
145 protocols using chemiluminescence
1
TIGAR Protein Expression in Colorectal Cancer
2
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor cells were lysed in 50 µl of RIPA buffer (Cell Signaling Technology, Inc.) with 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). The concentration of protein was measured using a BCA Protein Assay kit (Sigma-Aldrich; Merck KGaA). Whole-cell lysates were prepared, fractioned and separated (40 µg/lane) via SDS-PAGE (10% gel). The separated proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore) and were blocked with blocking buffer containing 5% non-fat milk in Tris-buffered saline and 0.1% Tween 20 (TBS-T) at 37°C for 2 h. The membranes were subsequently incubated with primary antibodies against E-cadherin (cat. no. ab15148), vimentin (cat. no. ab92547), GAPDH (cat. no. ab8245) or eIF5A2 (cat. no. ab150439) (all 1:1,000; all from Abcam) overnight at 4°C. The membranes were washed three times with TBST and then incubated with the appropriate horseradish-peroxidase-conjugated secondary antibodies (1:2,000; cat. no. ab7090; Abcam) for 1 h at room temperature. Protein expression was detected by chemiluminescence (GE Healthcare) and visualized by autoradiography on X-ray films.
3
Western Blot Analysis of Nuclear and Cytoplasmic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear and cytoplasmic extracts were prepared from liver tissue and cultured cells using a commercially available nuclear extraction kit (Cayman Chemical Co., Ann Arbor, MI, USA). Twenty micrograms of protein was resolved on a 4%-12% Bis-Tris NuPAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot Transfer Stack (Invitrogen). After protein transfer, the following primary antibodies were used: anti-phospho-GSK-3β (#9323; Cell Signaling Technology, Danvers, MA, USA), anti-GSK-3β (#9315; Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (#9582; Cell Signaling Technology, Danvers, MA, USA), anti-TCF4 (#2569; Cell Signaling Technology), anti-PPARγ (#2443; Cell Signaling Technology), anti-SREBP-1 (sc-366; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-β-actin (#4967; Cell Signaling Technology), and anti-Lamin B1 (ab16048; Abcam, Cambridge, MA, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
4
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and scraped in RIPA buffer containing 10% protease inhibitor cocktail to obtain the total protein content. The BCA method was used to measure the protein concentration. Each sample was fractionated using 10% SDS-PAGE and blotted onto PVDF membranes. The membranes were incubated in 5% nonfat dry milk to block nonspecific binding and then blotted with a primary antibody overnight at 4°C. After washes with TBST and incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Biosynthesis Biotechnology, China) for 2 h at room temperature, the immunocomplexes were visualized using chemiluminescence (GE, USA) according to the manufacturer’s protocol (Sun et al., 2008 (link)).
5
Immunoblotting Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bladder cancer cells were collected and lysed in 50 μL cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitors (Sigma-Aldrich). The protein concentration was quantified using a BCA Protein Kit (Thermo Fisher Scientific, Rockford, IL, USA). The cell lysates were separated by 10% SDS-PAGE and the proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA), blocked with TBS/T containing 5% BSA, and then incubated with primary antibodies against E-cadherin, vimentin, Twist-1, Zeb-1, snail, or eIF5A2 (Abcam, Cambridge, MA, USA) at 4°C overnight. The membranes were washed three times with TBS/T and then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. The protein bands were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA) and visualized by autoradiography (Kodak, Rochester, NY, USA).
6
Western Blot Analysis of PI3K/Akt/mTOR Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS). 10% SDS-polyacrylamide gel electrophoresis (PAGE) was implmented to separate the proteins. We used the polyvinylidene difluoride (PVDF; Tiangen) membranes in current study. Primary antibodies against p85 (a subunit of PI3K), p-p85 (Tyr458), Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2448) or GAPDH were purchased from Tiangen. After being washed with TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Tiangen). Chemi-luminescence was used to visualize blots (GE Healthcare, Fairfield, CT, USA).
7
Immunoblot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblots were performed as previously described [17 (link)]. Briefly, cells were lysed in lysis buffer (35 mM Tris [pH 7.4], 0.4 mM EGTA, 10 mM MgCl2, and 0.1% Triton-100) containing protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich). The total cell lysate was homogenized in 2× sodium dodecyl sulfate (SDS) sample buffer, boiled, subjected to SDS-polyacrylamide (10%) gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 1% BSA and incubated with the primary antibodies. After it was rinsed with 0.1% Tween-20 in PBS, the membrane was incubated with the appropriate HRP-conjugated secondary antibody. The intensity of the positive signals was visualized by chemiluminescence (GE Healthcare, Buckinghamshire, UK), and the images were imported by Image Reader LAS-1000 Plus (Fuji Photo Film Co. Ltd., Tokyo, Japan).
8
Western Blot Analysis of Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Astrocytes were washed in phosphate-buffered saline (PBS) and collected by gentle scraping in ice-cold RIPA buffer containing, in mM: 50 Tris pH 7.4, 100 NaCl, 1 Ethylene glycol tetraacetic acid (EGTA), 1 phenylmethylsulfonyl fluoride (PMSF), 1 sodium orthovanadate, 1 NaF, 0.5% NP-40, and 0.2% Sodium dodecyl sulfate (SDS) supplemented with protease inhibitor cocktail II [65 (link)]. Then, protein samples were separated on 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were incubated with rabbit polyclonal anti-KV3.4 (1:1000), rabbit polyclonal anti-caspase 12 (1:1000), rabbit polyclonal anti-GRP78/BiP (1:1000), and mouse monoclonal anti-β-actin (1:1000) antibodies. Immunoreactive bands were detected by chemiluminescence (GE Healthcare, Milan, Italy), and the software ImageJ (NIH, Bethesda, MD, USA) was used for densitometric analysis.
9
Western Blot Analysis of AQP4 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples (40 μg/lane) were separated using 10% SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with Tris-buffered saline (TBS) and 0.1% Tween-20 (TBS/T) containing 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies against AQP4 (Abcam, Cambridge, MA USA) diluted 1:1000 in TBS/T overnight at 4 °C. The membranes were washed and incubated with a horseradish peroxidase-labeled secondary antibody diluted 1:2000 at room temperature for 2 h. Subsequently, the protein bands were assessed using chemiluminescence (GE Healthcare, Piscataway, NJ, USA). The immunoreactive protein bands were quantified using Image Lab 5.0 (Bio-Rad, Hercules, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
10
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HCC cells lysates were resuspended in cell lysis buffer (Cell Signaling) and the protein concentrations were quantified using BCA Protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). The prepared protein lysates added with loading buffer and denatured by boiling were separated in 10% SDS-PAGE gels, and then transferred to Polyvinylidene Fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then PVDF membranes were incubated with relevant primary antibodies at 4 °C overnight. Then the membranes were incubated with the appropriate HRP-conjugated secondary antibody for 2 h. Protein bands were developed by chemiluminescence (GE Healthcare, Piscataway, NJ, USA) and visualized using an autoradiography kit (Kodak, Rochester, NY, USA).
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!