All 15 cell lines were examined for protein expression of p21 and p53 after treatment with Nutlin-3a at the IC50 dose via Western blot analysis. Protein lysates from cell cultures were extracted with radioimmunoprecipitation assay (RIPA) buffer and quantified by Bradford method. Electrophoresis of lysates (10 μg) was carried out on a 10% sodium dodecyl sulfate-polyacrylamide gel, followed by electroblot transfer onto a PVDF membrane. After blocking in 5% nonfat milk in phosphate-buffered saline (PBS) for 1 h, the membranes were probed with the following primary antibodies: Anti-mouse p21 (1:500 dilution; BD Pharmingen, San Diego, CA), anti-mouse p53 (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and β-actin (1:50000 dilution; Sigma-Aldrich, St. Louis, MO) were dissolved in PBS with 5% bovine serum albumin (BSA), added to the Western blots, and incubated overnight at 4°C. The blots were then rinsed and incubated with IR-dye 680–conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE). Membranes were then imaged using the LI-COR Odyssey Infrared Image Detection System (LI-COR Biosciences, Lincoln, NE) at 700 nm and 800 nm.
Irdye680 conjugated secondary antibodies
Manufactured by LI COR
IRDye680-conjugated secondary antibodies are fluorescent-labeled reagents designed for detection and quantification in western blotting and other immunoassay applications. The IRDye680 fluorescent dye is covalently attached to the secondary antibody, providing a stable conjugate for sensitive and specific target detection.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (3 mentions)
Irdye 800cw, by LI COR (2 mentions)
β actin, by Merck Group (1 mentions)
Cyp2e1, by Abcam (1 mentions)
Image studio ver 4, by LI COR (1 mentions)
Odyssey system, by LI COR (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Immobilon fl polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Rabbit anti human e2f1, by Santa Cruz Biotechnology (1 mentions)
Mouse anti human rrm2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti human creb1, by Santa Cruz Biotechnology (1 mentions)
Mouse anti human gapdh, by Santa Cruz Biotechnology (1 mentions)
Odyssey application software, by LI COR (1 mentions)
Mcherry ab167453, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
β actin, by LI COR (1 mentions)
Ab8245, by Abcam (1 mentions)
6 protocols using irdye680 conjugated secondary antibodies
1
Evaluating Nutlin-3a's Effects on p21 and p53
2
Quantitative Liver Protein Analysis
3
Quantifying FN splice variant expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the content of the FN EDA splice variant, adult human FN (ScienCell 8488) and newborn human FN (Sigma F2518) proteins underwent reduction and electrophoresis onto a 3 to 8% tris-acetate gradient gel (NuPAGE) and blotting onto Immobilon-FL polyvinylidene difluoride membrane (Millipore). The membrane was probed with rabbit anti-FN (Abcam) and mouse anti–EDA-FN (Ist-9) antibodies followed by IRDye 800CW– or IRDye 680–conjugated secondary antibodies (LI-COR Biosciences), and immunofluorescence was detected using the Odyssey Infrared Imaging System (LI-COR Biosciences). Pull-down assays were undertaken by coincubating 10 μg of adult or newborn FN with or without 5 μg of recombinant human SFRP4 (R&D Systems) in Dulbecco’s PBS containing Mg2+, Ca2+, 0.1% CHAPS, 0.1% octylglucoside, BSA (1 mg/ml), and 2% glycerol 4°C overnight then with rabbit anti-SFRP4 antibody loaded onto protein A agarose beads for 4 hours. Eluted proteins were run on a single gradient gel subsequently halved. Upper and lower halves were subjected to blotting conditions appropriate for transfer of either high or low molecular weight proteins, respectively. Blots were stained and read as described above. Relative protein densities were determined by ImageJ.
4
Whole Cell Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The whole cell lysate were analyzed with the antibodies mouse anti-human RRM2, rabbit anti-human CREB1, rabbit anti-human E2F1, mouse anti-human Flag, mouse anti-human cmyc, mouse anti-human GAPDH (Santa Cruz Biotechnology), IRDye® 800CW- or IRDye® 680-conjugated secondary antibodies (LI-COR, Lincoln,NE) were used for staining and then detected by an Odyssey® infrared imaging system (LI-COR).
5
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the detection of protein expression in total cell lysates, cells were lysed in Novex Tris-Glycine SDS sample buffer containing NuPAGE sample reducing agent (Invitrogen). Lysates were electrophoresed through Novex Tris-Glycine gels or NuPAGE Bis-Tris gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis. The blocking of membranes and subsequent antibody incubations were performed using Odyssey blocking buffer (LI-COR Biosciences) according to the manufacturer’s instructions. Primary antibodies against β-Tubulin (926–42211, LI-COR Biosciences), β-actin (926–42210, LI-COR Biosciences), FLAG (F1804, Millipore Sigma, Burlington, MA), mCherry (ab167453, Abcam, Cambridge, United Kingdom), GAPDH (ab8245, Abcam), and human SOD1 (ADI-SOD1–100, Enzo) were purchased from commercial sources. The IRDye 800CW-conjugated and IRDye 680-conjugated secondary antibodies were obtained from LI-COR Biosciences. Immunoblot signals were visualized by the Odyssey CLx infrared imaging system and quantified by ODYSSEY application software (LI-COR Biosciences).
6
Immunoprecipitation and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation and immunoblotting were carried out as previously described [22 (link),23 (link),49 (link)]. Briefly, cells were harvested, rinsed with ice-cold PBS, and lysed with NP40 buffer (50 mM Tris-HCL [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% NP40) supplemented with protease inhibitor cocktail. Centrifuged cell lysates were then pre-cleared with Sepharose 4B beads, and subjected to precipitation with antibody-conjugated agarose (Sigma) at 4°C for 4–6 hours. Precipitated proteins were extensively washed with NP40 buffer and eluted with 1x SDS-PAGE loading buffer by boiling at 95°C for 5–10 minutes.
For immunoblotting analysis, whole cell lysates (WCL) or precipitated proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membrane. Immunoblotting analysis was performed with indicated primary antibodies and proteins were visualized with IRDye800- or IRDye680-conjugated secondary antibodies (Licor) using an Odyssey infrared imaging system (Licor).
For immunoblotting analysis, whole cell lysates (WCL) or precipitated proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membrane. Immunoblotting analysis was performed with indicated primary antibodies and proteins were visualized with IRDye800- or IRDye680-conjugated secondary antibodies (Licor) using an Odyssey infrared imaging system (Licor).
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!