All immunoblot samples were heated at 100°C for 10 min. Equal amounts of protein were separated by SDS‐polyacrylamide gel electrophoresis (EZBiolab) and transferred to nitrocellulose membranes (Pall Corporation). The membranes were blocked with 5% non‐fat milk diluted in PBS with 0.1% Tween 20 (PBST) for 1 h and then incubated at 4°C overnight with primary antibodies. The membranes were washed with PBST three times and incubated with secondary antibodies for 1 h at room temperature followed by washing again with PBST three times. Finally, enhanced chemiluminescence imaging was performed on a chemiluminescent imaging system (5200, Tanon). Quantitation of immunoblot results was performed with the ImageJ software (National Institutes of Health). The following primary antibodies were used for immunoblotting: antibodies against HA Tag (3724, CST), HuR (2582, CST), and pERK1/2 (4370, CST) were used at 1:1,000 dilution, and antibodies against Vinculin (13901, CST) and Tubulin (T6199, Sigma) were used at 1:3,000 dilution as the internal controls. The secondary antibodies were goat anti‐rabbit IgG (1:5,000 dilution, M21001L, Abmart) or goat anti‐mouse IgG (1:5,000 dilution, M21002L, Abmart) conjugated to horseradish peroxidase.
Nitrocellulose membrane
Manufactured by Pall Corporation
Sourced in United States, China, Germany
Nitrocellulose membranes are a type of lab equipment used for various applications in scientific research and analysis. These membranes are made from a derivative of cellulose and are designed to facilitate the transfer and immobilization of biomolecules, such as proteins and nucleic acids, for further analysis or detection. The primary function of nitrocellulose membranes is to provide a stable and efficient platform for these biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (17 mentions)
Ripa buffer, by Beyotime (11 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
β actin, by Merck Group (7 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Bca kit, by Beyotime (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Quantity one software, by Bio-Rad (5 mentions)
Protease inhibitor, by Roche (5 mentions)
Las 4000 system, by Fujifilm (4 mentions)
Protease inhibitor, by Beyotime (4 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (4 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (4 mentions)
Gapdh, by Merck Group (4 mentions)
Cell lysis buffer, by Cell Signaling Technology (4 mentions)
251 protocols using nitrocellulose membrane
1
Immunoblot Analysis of Protein Targets
2
Protein Expression Quantification by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared using an ice-cold PRO-PREP protein extraction solution (iNtRON Biotechnology Inc., Seongnam, Korea). For all samples, 20 μg protein lysates was separated on SDS–polyacrylamide gels, transferred onto nitrocellulose membranes (Pall Corporation, NY, USA), and blocked with 5% skim milk. The samples were analyzed, as per the manufacturer instructions, using the following primary antibodies: Twist1 (Cell Signaling Biotechnology), PRX6 and Vimentin (AbFrontier, Seoul, Korea), E-cadherin (BD Biosciences, NJ, USA), and Snail and GAPDH (Santa Cruz Biotechnology Inc.). After washing off the excess secondary antibodies (horseradish peroxidase-conjugated goat antirabbit, mouse, and donkey antigoat, obtained from Thermo Scientific, IL, USA), specific banding was detected using Clarity Western ECL Substrate (Bio-Rad, CA, USA).
3
Western Blot Analysis of Sinonasal Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses were performed using sinonasal tissue extracts, prepared from samples collected during sinus surgeries at USF. For immunoblotting, the proteins were separated on 8%–12% SDS-PAGE and transferred onto nitrocellulose membranes (Pall Corporation, Washington, NY, United States). After blocking, primary antibodies (anti-MUC7 and anti-LPO) were diluted according to the manufacturer’s instructions. The blotted proteins were detected with an enhanced chemiluminescence (ECL) detection system (Thermo Scientific Pierce, Rockford, IL, United States). GAPDH was used as a loading control.
4
KYC4048 Metabolites Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at 2 × 105 cells/ml into 6-cm culture dishes, cultured for 1 day, and treated with 100 μg/ml non-polar, ethyl acetate, or n-hexane fractions (20 μg/ml, respectively) of KYC4048 metabolites for 24, 48, and 72 h. These cells were then washed with ice-cold PBS and lysed with a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 0.5% protease inhibitor cocktail. The cell lysates were separated by SDS-PAGE and the proteins were transferred to nitrocellulose membranes (Pall Corporation). The membranes were then blocked with a buffer (Tris-buffered saline/Tween-20 containing 5% skim milk) for 1h at room temperature followed by incubation with the indicated primary antibodies overnight at 4°C. The next day, the membranes were labeled with a secondary antibody. Immunoblots were visualized using an enhanced ECL kit (Santa Cruz Biotechnology).
5
Western Blotting Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular protein was prepared using a cell lysis buffer (Intron, Daejeon, Korea). Sample was run onto sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% skim milk, the membrane was incubated with primary antibody and peroxidase-conjugated secondary antibody sequentially. The Western band was visualized using enhanced chemiluminescence (Intron).
6
Quantification of Phosphorylated AMPK in Human Monocyte-Derived Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described [19] . Briefly, human mo-DCs were lysed in a buffer (1 mM each: antipain, benzamidine, leupeptin, pepstatin A, and phenylmethyl sulfonyl fluoride (PMSF), with 1% sodium dodecyl sulphate (SDS), and 5 mM ethylene diamine tetraacetic acid (EDTA)) to obtain the total protein. Proteins (30 µg protein/lane) were electrophoresed on an 8% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Pall Corporation, Ann Arbor, MI). After being blocked with BSA (3% wt/vol) in tris-buffered saline containing Tween 20 (0.5% vol/vol; TBS-T) for 1 h at room temperature, the membranes were incubated with antibodies against p-AMPK (1:1000), AMPK (1:1000) and tubulin (1:3000) overnight at 4°C. After rinsing three times, the blots were then incubated with the corresponding secondary antibodies at room temperature for 1 h. Signals of bands were visualized by an enhanced chemiluminescence (ECL) detection system (Bio-Rad, Hercules, CA) and quantified using Quantity One Software (Bio-Rad).
7
Western Blot Analysis of Immunotherapeutic Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular protein was collected with 2 × SDS sample buffer and subsequently denatured for 3 min at 95 °C. The protein samples were separated in 10% Nu-PAGE Bis–Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After being blocked with 5% (w/v) milk for 1 h, the membranes were incubated with the following antibodies: rabbit anti-PD-L1 antibody (#28076, 1:2000, Proteintech, Wuhan, China), mouse anti-β-actin antibody (#sc-47778, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, USA), rabbit anti-GSDME antibody (#ab215191, 1:2000, Abcam, Cambridge, UK, USA), and mouse anti-GAPDH (#sc-47724, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA).
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed, as previously described42 (link), and separated using SDS-PAGE followed by transfer to nitrocellulose membranes (Pall Corporation, Port Washington, NY). Membranes were probed at 4 °C overnight with the appropriate primary antibodies followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized by chemiluminescence (Western blotting luminol reagent; Santa Cruz, Dallas, TX) using Licor Odyssey Fc imaging system.
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A small amount of minced tissue block was placed in a 2-mL Eppendorf tube, and cleaned steel beads were added. Then, 300 μL of detergent lysate containing phenylmethylsulfonyl fluoride were added to each tube, which was placed in an automatic homogenizer for homogenization. After incubation on ice for 30 minutes, the supernatant was collected. After determining the protein concentration, samples were denatured, and 40 µg of protein were loaded into each well and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA), and the membranes were then blocked with 5% non-fat milk in Tris-buffered saline/Tween 20 for 2 hours at room temperature. Primary antibodies (all from Affinity, Shanghai, China) against GAPDH (1:1000), MYPT1 (1:1000), p-MYPT1 (1:1000), ROCK1 (1:1000), and ROCK2 (1:1000) were diluted with blocking solution, and the membranes were incubated in the primary antibody overnight at 4°C. The membranes were thoroughly washed with Tris-buffered saline/Tween 20 five times for 5 minutes each. The membranes were soaked in secondary antibody (1:600) for 2 hours at room temperature on a shaker. The membranes were again thoroughly washed with Tris-buffered saline/Tween 20 five times for 5 minutes each. The film was developed and then scanned.
10
Western Blot Analysis of RCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of RCC cells was extracted with RIPA lysis buffer with fresh protease and phosphatase inhibitor cocktail. A bicinchoninic acid (BCA) Kit (Beyotime Biotechnology) was used to detect protein concentration. Next, 20 μg protein samples were electrophoresed by 7.5% SDS–PAGE and then transferred to nitrocellulose membranes (Pall Corporation). After blockade in 5% skim milk for 1 h at room temperature, the membranes were incubated at 4 °C overnight with the corresponding antibodies. The next day, the membranes were rinsed with washing buffer and then incubated with secondary antibodies. After rewashing the membranes, the signal was detected by a sensitive chemiluminescence detection system.
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!