Ab6046

Manufactured by Cell Signaling Technology

Sourced in United Kingdom

Ab6046 is a primary antibody product offered by Cell Signaling Technology. It is designed for use in various research applications.

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Lab products found in correlation

4 protocols using ab6046

Comprehensive Western Blot and Immunoprecipitation Protocol

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Western blot analysis was performed with lysates from cells or tissues
with Urea buffer (8 M Urea, 1 M Thiourea, 0.5% CHAPS, 50 mM DTT, and 24 mM
Spermine). For immunoprecipitations, the cells were lysed for 30 min at
4°C in pNAS buffer (50 mMTris-HCl [pH 7.5], 120 mM NaCl, 1 mM EDTA, and
0.1% Nonidet P-40), with protease inhibitors. Indicated antibodies and
immunoglobulin G (IgG) agarose were added to the lysate for 16 hours at
4°C. Immunoprecipitates were washed four times with cold PBS followed by
the addition of SDS sample buffer. The bound proteins were separated on SDS
polyacrylamide gels and subjected to immunoblotting with the indicated
antibodies. Primary antibodies were from: Abcam (SGLT1, 1:1000, ab14686;
β-actin, 1:2000, ab8227; β-tubulin, 1:5000, ab6046; GAPDH, 1:2000,
ab9385; Phospho-EGFR^Tyr1068, 1:1000, ab40815), Cell Signaling
Technology (SGLT1, 1:1000, 5042; Phospho-EGFR^Tyr1068, 1:1000, 3777,
D7A5; EGFR, 1:1000, 4267, D38B1; Phospho-AKT Ser473, 1:1000, 9271; Phospho-AKT
Thr308, 1:1000, 4056, 244F9; AKT, 1:1000, 9272; Phospho-ERK (Thr202/Tyr204),
1:1000, 9101; ERK, 1:1000, 9102; Cleaved PARP Asp214, 1:1000, 9541), Millipore
(PTEN, 1:1000, 04-409). Signals were detected using an ECL detection system (GE
Healthcare) or an Odyssey imaging system (LI-COR), and evaluated by ImageJ 1.42q
software (National Institutes of Health).

Liu H., Ertay A., Peng P., Li J., Liu D., Xiong H., Zou Y., Qiu H., Hancock D., Yuan X., Huang W., Ewing R.M., Downward J, & Wang Y. (2019). SGLT1 is required for the survival of triple‐negative breast cancer cells via potentiation of EGFR activity. Molecular Oncology, 13(9), 1874-1886.

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Immunoprecipitation and Western Blot Analysis

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Western blot analysis was performed with lysates from cells or tissues with urea buffer (8 m urea, 1 m thiourea, 0.5% CHAPS, 50 mm DTT and 24 mm spermine). For immunoprecipitations, the cells were lysed for 30 min at 4 °C in pNAS buffer [50 mm Tris/HCl (pH 7.5), 120 mm NaCl, 1 mm EDTA and 0.1% Nonidet P-40], with protease inhibitors. Indicated antibodies and immunoglobulin G (IgG) agarose were added to the lysate for 16 h at 4 °C. Immunoprecipitates were washed four times with cold PBS followed by the addition of SDS sample buffer. The bound proteins were separated on SDS polyacrylamide gels and subjected to immunoblotting with the indicated antibodies. Primary antibodies were from: Sigma (ASPP1, HPA006394; ASPP2, HPA021603), Santa Cruz Biotechnology (ZEB1, sc-25388; E-cadherin, sc-21791; Snail2, sc-166476), Abcam (β-tubulin, ab6046; NF-κB1 p50, ab32360; NF-κB2 p52, ab174482; ZEB2, ab138222), Cell Signaling Technology (phospho-AKT, 9271; AKT, 4685; Snail1, 3879; Snail2, 9585; NF-κB p65, 8242), BD Transduction Laboratories (E-cadherin, 610405). Signals were detected using an ECL detection system (GE Healthcare) (Chicago, IL, USA) or an Odyssey imaging system (LI-COR), and evaluated by ImageJ 1.42q software (National Institutes of Health) (Berhesda, MD, USA).

Liu D., Ertay A., Hill C., Zhou Y., Li J., Zou Y., Qiu H., Yuan X., Ewing R.M., Lu X., Xiong H, & Wang Y. (2020). ASPP1 deficiency promotes epithelial-mesenchymal transition, invasion and metastasis in colorectal cancer. Cell Death & Disease, 11(4), 224.

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Immunoblot and Immunofluorescence Antibody Protocols

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The following antibodies were used for immunoblot analysis: rabbit-anti-IKK2 (CST-2678, Cell Signaling, Danvers, MA, USA), rabbit-anti-p-p65 (CST-3033, Cell Signaling, Danvers, MA, USA), rabbit-anti-p65 (sc-372, Dallas, TX, USA), chicken-anti-GFP (ab13970, Cambridge, UK), rabbit-anti-tubulin (ab6046, Cambridge, UK), rabbit-anti-PLP1 (CST-28702, Cell Signaling, Danvers, MA, USA), rabbit-anti-MOG (CST-96457, Cell Signaling, Danvers, MA, USA), rabbit-anti-MBP (CST-2396, Cell Signaling, Danvers, MA, USA), rabbit-anti-GAPDH (CST-3686, Cell Signaling, Danvers, MA, USA), rabbit-anti-p-eIF2α (CST-3597, Cell Signaling, Danvers, MA, USA), rabbit-anti-eIF2α (CST-5324, Cell Signaling, Danvers, MA, USA), rabbit-anti-ERK2 (sc-1647, Dallas, TX, USA).
For immunofluorescence, the following primary antibodies were used: mouse-anti-CC1 (OP80, Merck, Darmstadt, Germany), mouse-anti-GSTπ (BD610719, BD, Franklin Lakes, NJ, USA), chicken-anti-GFP (GFP-1020, AvesLab, Davis, CA, USA), mouse-anti-GFAP (sc-33673, Dallas, TX, USA), rabbit-anti-NG2 (AB5320, Merck, Darmstadt, Germany), rabbit-anti-MBP (Biolegend 836,504, San Diego, CA, USA), mouse-anti-Neurofilament-H (SMI32P) (Biolegend 801,701, San Diego, CA, USA), rabbit-anti-ß3-tubulin (Biolegend 802,001, San Diego, CA, USA), rabbit- anti RFP (abcam ab124754, Cambridge, UK).

Schlett J.S., Mettang M., Skaf A., Schweizer P., Errerd A., Mulugeta E.A., Hein T.M., Tsesmelis K., Tsesmelis M., Büttner U.F., Wendt H., Abaei A., Rasche V., Prex V., Nespoli E., Alami N.O., Tews D., Walther P., Yilmazer-Hanke D., Oswald F., Dimou L., Wirth T, & Baumann B. (2023). NF-κB is a critical mediator of post-mitotic senescence in oligodendrocytes and subsequent white matter loss. Molecular Neurodegeneration, 18, 24.

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Western Blot Analysis of Protein Samples

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Cell lysates were harvested by directly lysing the cells with 2 × sampling buffer (Bio-Rad). Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in TBS-Tween (TBST)
for 1 hour, incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish-peroxidase-conjugated secondary antibodies for 1 hour, and illuminated by enhanced chemiluminescence (ECL). Quantification of band intensity was done in Image J. -actin antibody: Abcam, Ab20272 (1:10,000); -tubulin antibody: Abcam, Ab6046 (1:5,000); H2B antibody: Cell Signaling, 8135 (1:2,000).

Hu X., Wu Q., Zhang J., Chen X., Hartman A.A., Eastman A.E., & Guo S. (2020). Reprogramming progressive cells display low CAG promoter activity.

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