Ab177149

Manufactured by Abcam

Sourced in United Kingdom

Ab177149 is a monoclonal antibody that recognizes the human RALBP1 protein. RALBP1 is a Ral binding protein that functions as a GTPase activating protein (GAP) for the Ral GTPases. The antibody can be used for detection of RALBP1 in various applications.

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Lab products found in correlation

β actin, by Merck Group (3 mentions) P54nrb nono, by Merck Group (2 mentions) Ab32157, by Abcam (2 mentions) B 5 1 2, by Merck Group (1 mentions) Odyssey clx infrared imaging system, by LI COR (1 mentions) Ab205718, by Abcam (1 mentions) Rt qpcr kit, by GeneCopoeia (1 mentions) Ab128915, by Abcam (1 mentions) Ab84078, by Abcam (1 mentions) G3bp1, by BD (1 mentions) Total eif2α, by Cell Signaling Technology (1 mentions) Pspc1, by Merck Group (1 mentions) Sc 9996, by Santa Cruz Biotechnology (1 mentions) Cpsf6, by Fortis Life Sciences (1 mentions) Pabpc1, by Cell Signaling Technology (1 mentions) Eif4e, by BD (1 mentions) Dazap1, by Fortis Life Sciences (1 mentions) Hnrnpk, by Fortis Life Sciences (1 mentions) Tdp 43, by Merck Group (1 mentions) β actin, by Cell Signaling Technology (1 mentions)

7 protocols using ab177149

Western Blot Analysis of Protein Targets

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Cells were lysed in 2× Laemmli sample buffer and were boiled for 5 min. Protein size-fractionation by a 6% SDS–PAGE gel and subsequent electro-transfer to a PVDF membrane (0.45 μm) was accomplished as described (36 (link)). Blotting was performed overnight at 4°C, 75 mA in 2× Blot-buffer (50 mM Tris, 384 mM Glycin, 0.02% SDS) without methanol. Blocking of the membranes was accomplished with 3% BSA in PBS for 1 h at room temperature. Membranes were then washed three times with PBS containing 0.05% Tween and were subsequently incubated with primary antibodies against CSB (E-18, sc-10459, Santa Cruz Biotechnology, Inc., 1:250 in PBS 3% BSA), Tubulin (B512, Sigma-Aldrich, 1:5000 in PBS 3% BSA), or OGG1 (ab12474, Abcam, 1:1000 in PBS 3% BSA) or SFPQ (ab177149, Abcam, 1:2000 in PBS 3% BSA) in combination with Odyssey-compatible secondary antibodies. Western blots were analyzed using the Odyssey CLx Infrared Imaging System (LI-COR Biosciences).

Menoni H., Wienholz F., Theil A.F., Janssens R.C., Lans H., Campalans A., Radicella J.P., Marteijn J.A, & Vermeulen W. (2018). The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage. Nucleic Acids Research, 46(15), 7747-7756.

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Quantifying NEAT1 and SFPQ Expression

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RT-qPCR of NEAT1_2 and SFPQ mRNA in tissue specimens or cell lines were performed using a custom-made RT-qPCR kit (GeneCopoeia, Maryland, USA), following the manufacturer’s instructions. The semi-quantitative 2^−ΔΔCt method was employed for qPCR data analysis, and expression levels of NEAT1_1, NEAT1_2 or SFPQ mRNA in each sample were normalized to that of GAPDH mRNA before further analysis. Western blotting of SFPQ in different cell cultures was performed using a rabbit anti-human SFPQ monoclonal antibody (ab177149), rabbit anti-human GAPDH monoclonal antibody (ab128915) and horseradish peroxidase-conjugated goat anti-rabbit second antibody (ab205718, Abcam, Cambridge, UK), following the manufacturer’s instructions. Western blotting results were further analyzed by normalizing the gray scale of the SFPQ band in each sample to that of GAPDH using ImageJ software before statistical analysis.

Ru Y., Chen X.J., Guo W.Z., Gao S.G., Qi Y.J., Chen P., Feng X.S, & Zhang S.J. (2018). NEAT1_2–SFPQ axis mediates cisplatin resistance in liver cancer cells in vitro. OncoTargets and therapy, 11, 5695-5702.

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Immunodetection of FUS and Related Proteins

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The following commercial primary antibodies were used: FUS full protein (rabbit polyclonal, 11,570–1-AP); FUS N-terminus (rabbit polyclonal, Abcam, ab84078; aa. 1–50); FUS C-terminus (Bethyl, A300-294A; aa. 500–526); p54nrb/NONO (rabbit polyclonal C-terminal, Sigma); SFPQ (rabbit monoclonal, ab177149, Abcam; rabbit polyclonal, A301-322A, Bethyl); beta-actin (mouse monoclonal, A5441, Sigma). Antibodies were used at 1:500–1:1000 dilution for all applications unless stated otherwise.

An H., Skelt L., Notaro A., Highley J.R., Fox A.H., La Bella V., Buchman V.L, & Shelkovnikova T.A. (2019). ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles. Acta Neuropathologica Communications, 7, 7.

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Comprehensive Antibody Panel for Protein Analysis

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The following commercial primary antibodies were used: G3BP1 (mouse monoclonal; BD Biosciences); TIAR (mouse monoclonal; BD Biosciences); NONO (rabbit polyclonal C-terminal; Sigma-Aldrich); SFPQ (rabbit monoclonal; ab177149, Abcam); CPSF6 (rabbit polyclonal; A301-356A, Bethyl); FUS (mouse monoclonal; 4H11, Santa Cruz); ELAVL1 (rabbit polyclonal, 11910-1-AP, Proteintech); PABPC1 (rabbit polyclonal, Cell Signaling, 4992); EIF4E (mouse monoclonal; BD Biosciences); UBAP2L (rabbit polyclonal; A300-533A, Bethyl); DAZAP1 (rabbit polyclonal; A303-984A, Bethyl); hnRNP K (rabbit polyclonal; A300-674A, Bethyl); PSPC1 (rabbit polyclonal N-terminal; Sigma-Aldrich); TDP-43 (rabbit polyclonal C-terminal; Sigma-Aldrich); HNRNPA3 (rabbit polyclonal; 25142-1-AP, Proteintech); RBM12B (rabbit polyclonal; 17137-1-AP, Proteintech); SRSF9 (rabbit polyclonal; 17926-1-AP, Proteintech); SMARCA5 (rabbit polyclonal; 13066-1-AP, Proteintech); MATR3 (rabbit polyclonal; 12202-2-AP, Proteintech); YBX1 (rabbit polyclonal; 20339-1-AP, Proteintech); GFP (mouse monoclonal; sc-9996, Santa Cruz); Flag (DYKDDDDK tag, mouse monoclonal; 9A3, Cell Signaling); eIF2α phosphorylated at Ser51 (rabbit monoclonal; ab32157, Abcam); total eIF2α (rabbit monoclonal; D7D3, Cell Signaling); and β-actin (mouse monoclonal; A5441, Sigma-Aldrich). Antibodies were used at 1:1,000 dilution for all applications unless stated otherwise.

An H., Tan J.T, & Shelkovnikova T.A. (2019). Stress granules regulate stress-induced paraspeckle assembly. The Journal of Cell Biology, 218(12), 4127-4140.

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Antibody Panel for Neurodegeneration Markers

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The following commercial primary antibodies were used: TDP-43 (rabbit polyclonal, 10782–2-AP, Proteintech and mouse monoclonal, MAB7778-SP, R&D Biosystems); FUS (rabbit polyclonal, Proteintech, 11570–1-AP); p54nrb/NONO (rabbit polyclonal C-terminal, Sigma); PSF/SFPQ (rabbit monoclonal, ab177149, Abcam); Tuj (β-Tubulin III, mouse monoclonal, Sigma); dsRNA (mouse monoclonal, J2, Kerafast); cleaved caspase 3 (rabbit polyclonal, 9661, Cell Signaling); NF-κB p65 (rabbit monoclonal, D14E12, Cell Signaling); IFIT3 (rabbit polyclonal, Bethyl); p-eIF2α (rabbit monoclonal, ab32157, Abcam); p-PKR (rabbit polyclonal, Thr451, ThermoFisher); PKR (mouse monoclonal, MAB1980-SP, R&D Systems); eIF2α (rabbit monoclonal, D7D3, Cell Signaling); β-actin (mouse monoclonal, A5441, Sigma). Antibodies were used at 1:500–1:1000 dilution for all applications.

Shelkovnikova T.A., Kukharsky M.S., An H., Dimasi P., Alexeeva S., Shabir O., Heath P.R, & Buchman V.L. (2018). Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis. Molecular Neurodegeneration, 13, 30.

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Protein Extraction and Western Blot

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Total cell lysates were prepared for western blot by adding 2× Laemmli buffer directly to the wells in a 24-well plate followed by denaturation at 100°C for 5 min. SDS-PAGE and detection of proteins were carried out as described elsewhere (26 (link)). The following commercial primary antibodies were used (1:1000 dilution): FUS (rabbit polyclonal, 11570-1-AP, Proteintech); NONO (rabbit polyclonal C-terminal, Sigma); SFPQ (rabbit monoclonal, ab177149, Abcam); beta-actin (mouse monoclonal, A5441, Sigma).

An H., Elvers K.T., Gillespie J.A., Jones K., Atack J.R., Grubisha O, & Shelkovnikova T.A. (2022). A toolkit for the identification of NEAT1_2/paraspeckle modulators. Nucleic Acids Research, 50(20), e119.

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Western Blot Analysis of Signaling Proteins

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Equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies, including rabbit anti-ILF3 (1:2000, ab93255, Abcam), rabbit anti-SFPQ (1:2000, ab133574, Abcam), rabbit anti-NONO (1:2000, ab177149, Abcam), rabbit anti-GAPDH (1:5000, 5174, Cell Signaling Technology), rabbit anti-H3 (1:5000, 9728, Cell Signaling Technology), rabbit anti-SOCS2 (1:2000, ab109245, Abcam), rabbit anti-pJAK2 (1:2000, ab32101, Abcam), rabbit anti-JAK2 (1:2000, ab108596, Abcam), rabbit anti-pSTAT5 (1:2000, ab32364, Abcam), rabbit anti-STAT5 (1:2000, ab200341, Abcam), rabbit anti-pSTAT3 (1:2000, ab76315, Abcam), rabbit anti-STAT3 (1:2000, ab68153, Abcam). HRP-linked secondary antibody was used as the secondary antibody. Signals were visualized with chemiluminescence (Millipore, MA, USA).

Wang S., Han H., Meng J., Yang W., Lv Y, & Wen X. (2021). Long non-coding RNA SNHG1 suppresses cell migration and invasion and upregulates SOCS2 in human gastric carcinoma. Biochemistry and Biophysics Reports, 27, 101052.

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