Primary abs

Manufactured by Cell Signaling Technology

Sourced in United States

Primary Abs are the essential tools used in immunological research and analysis. They are antibodies that specifically bind to target proteins or antigens, allowing researchers to detect and quantify the presence of these biomolecules within a sample. Primary Abs serve as the foundation for various immunoassay techniques, enabling the identification and study of cellular signaling pathways, protein expression patterns, and other fundamental biological processes.

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5 protocols using primary abs

Protein Expression Analysis in Cells and Tumors

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Rabbit monoclonal Abs against p-Akt (#4060), total forms of Akt (#2938), p-mTOR (#5536), mTOR (#2972), p-4EBP1 (#9451), 4E-BP1 (#9452), PAK1 (#2602), HA tag (#3714), Atg14 (#96752), Vps34 (#4263), PI3K (#4249), LC3A/B (#4108), Beclin1 (#3495), and Atg5 (#12994) were purchased from Cell Signaling Technology. Mouse monoclonal Abs against ubiquitin (sc-271289) were purchased from Santa Cruz Biotechnology.
The samples derived from cells and tumor homogenates were lysed in Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), separated by electrophoresis on 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose transfer membranes (Merck Millipore, Cork, Ireland). Proteins were detected using primary Abs at a concentration of 1:1,000 (Cell Signaling Technology) and were incubated overnight. Specific interaction with the primary Abs was detected using corresponding secondary Abs conjugated to HRP (Biosharp), and signals were developed using the enhanced chemiluminescence (ECL) reagents (Biosharp). Gel bands were quantified by ImageJ software, and data are presented as mean ± SD from three independent immunoblotting assays.⁵¹ (link) Phosphorylated and total protein levels were determined and quantified by three successive immunoblotting membranes.

Bu H., Tan S., Yuan B., Huang X., Jiang J., Wu Y., Jiang J, & Li R. (2020). Therapeutic potential of IBP as an autophagy inducer for treating lung cancer via blocking PAK1/Akt/mTOR signaling. Molecular Therapy Oncolytics, 20, 82-93.

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Protein Extraction and Western Blot Analysis

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Detailed assays were carried out as previously reported.¹⁶, ¹⁷, ¹⁸ Briefly, RIPA lysis buffer (Invitrogen) was used to extract total proteins. After examining the protein concentration by BCA Kit (Beyotime Technology), 10% SDS‐PAGE was isolated the proteins and transferred to PVDF membranes. After blocking with 5% milk, membranes were cultured in primary Abs (Cell Signaling Technology) and subsequently probed with secondary Ab. Then the signals were determined using a chemiluminescence system (Thermo Fisher Scientific).

Liu Z., Mo H., Sun L., Wang L., Chen T., Yao B., Liu R., Niu Y., Tu K., Xu Q, & Yang N. (2020). Long noncoding RNA PICSAR/miR‐588/EIF6 axis regulates tumorigenesis of hepatocellular carcinoma by activating PI3K/AKT/mTOR signaling pathway. Cancer Science, 111(11), 4118-4128.

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Phosphorylation of C-Jun and NF-κB in THP-1 Cells

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THP-1 cells were harvested and lysed by incubation for 30 min with lysis buffer containing Tris (62.5 mM; pH 7.5), 1% Triton X-100, and 10% glycerol. Supernatants were collected by centrifuging cell lysates at 14,000 rpm for 10 min, and protein concentration was measured by using Quick Start Bradford-13 Dye Reagent and Bio-Rad protein assay kit (Hercules, CA, USA). Samples (20 μg) were mixed with loading buffer, heated at 95 °C for 5 min, and resolved by 12% SDS-PAGE. Fractionated proteins were electroblotted to an Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, USA) for transfer, blocked for 1h using 5% nonfat milk in PBS, and incubated at 4 °C overnight with primary Abs (1:100 dilution; Cell Signaling Technology Inc., Danvers, MA, USA) against p-C-Jun and p-NF-κB. Blots were washed four times with Tris buffered saline with 0.1% Tween 20 and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA). Immunoreactive bands were developed using Amersham ECL Plus Western Blotting Detection System (GE Health Care, Buckinghamshire, UK) and visualized by Molecular Imager® VersaDoc™ MP Imaging Systems (Bio-Rad Laboratories, Irvine, CA, USA).

Sindhu S., Akhter N., Wilson A., Thomas R., Arefanian H., Al Madhoun A., Al-Mulla F, & Ahmad R. (2020). MIP-1α Expression Induced by Co-Stimulation of Human Monocytic Cells with Palmitate and TNF-α Involves the TLR4-IRF3 Pathway and Is Amplified by Oxidative Stress. Cells, 9(8), 1799.

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Immunodetection of Cell Cycle Regulators

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The method used was described earlier [6 (link)]. Primary Abs obtained from Cell Signaling (Danvers, MA, USA) was used in the following dilutions: anti-p16^INK4A rabbit Ab (1:800 dilution), anti-p21^Waf1/Cip1 rabbit antibody (1:400 dilution).

Rzepka Z., Rok J., Kowalska J., Banach K, & Wrześniok D. (2022). Cobalamin Deficiency May Induce Astrosenescence—An In Vitro Study. Cells, 11(21), 3408.

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Quantification of Secreted Factors and Protein Analysis

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Secreted factors in tissue culture supernatants and sera were quantified by ELISA according to manufacturer’s instructions (eBioscience) or by using Bio-Plex bead capture assays and a Bio-Plex 200 analyzer (Bio-Rad). Cell lysates were resolved on SDS-PAGE gels and probed with Primary Abs, and immunoreactive proteins were visualized with HRP-coupled secondary Abs and chemiluminescence reagents (Bio-Rad, Pierce, or Cell Signaling Technology). Primary Abs were from Cell Signaling Technology, with the exception of an anti-TTP antiserum that was described previously (40 (link)). Blots were visualized using the ChemiDoc MP Imaging System, and in some cases quantified using Image Lab software (Bio-Rad).

Smallie T., Ross E.A., Ammit A.J., Cunliffe H.E., Tang T., Rosner D.R., Ridley M.L., Buckley C.D., Saklatvala J., Dean J.L, & Clark A.R. (2015). Dual-Specificity Phosphatase 1 and Tristetraprolin Cooperate To Regulate Macrophage Responses to Lipopolysaccharide. The Journal of Immunology Author Choice, 195(1), 277-288.

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