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Anti sam68

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Anti-Sam68 is a laboratory reagent used to detect and quantify the expression of the Sam68 protein in biological samples. Sam68 is an RNA-binding protein involved in various cellular processes. This antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to study the expression and localization of Sam68 in research applications.

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Lab products found in correlation

Rnase 1, by Thermo Fisher Scientific (2 mentions) Rnase inhibitor, by Promega (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Anti iκbα, by Santa Cruz Biotechnology (1 mentions) Anti β actin, by Merck Group (1 mentions) Anti tubulin, by Merck Group (1 mentions) Anti gapdh, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Anti ahr, by Enzo Life Sciences (1 mentions) Anti nucleolin, by Abcam (1 mentions) Anti hnrnp c1 c2, by Santa Cruz Biotechnology (1 mentions) Anti nup62, by BD (1 mentions) Anti pabp, by Cell Signaling Technology (1 mentions) Anti sc35, by Merck Group (1 mentions) Anti nup153, by Abcam (1 mentions) Anti α β tubulin, by Cell Signaling Technology (1 mentions) Rnase free dnase, by Thermo Fisher Scientific (1 mentions) Anti hnrnp a1, by Santa Cruz Biotechnology (1 mentions) Anti ki67, by Abcam (1 mentions) Protein g magnetic dynabeads, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Sam68 (8 citations) Anti-Sam68 (C-20) (4 citations) Rabbit anti-Sam68 (4 citations)

8 protocols using anti sam68

1

Cytoplasmic and Nuclear Protein Fractionation

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10–20 × 106 cells were incubated in cytoplasmic lysis buffer (HEPES pH 7.6 10 mM, EGTA 0.1 mM, KCl 10 mM, MgCl2 1.5 mM, dithiothreitol 1 mM, NaF 20 mM, protease inhibitor cocktail (Roche), PMSF 1 mM) for 10 min at 4°C. Detergent NP‐40 was added at a final concentration of 0.2%, and cells were incubated for 1 min on ice. Supernatant was collected as cytoplasmic protein fraction. The nuclei pellet was added with nuclear lysis buffer (Tris–HCl pH 7.5 50 mM, NaCl 150 mM, EDTA 2 mM, Triton X‐100 1%, dithiothreitol 5 mM, deoxycholate 0.5%, SDS 0.1%, protease inhibitor cocktail, PMSF 1 mM) and incubated 15 min at 4°C. Supernatant was collected as nuclear protein fraction.
For Western blot, following antibodies were used: anti‐AhR 1:1,000 (Enzo Life Sciences, BML‐SA210); anti‐Sam68 1:2,000 (Santa Cruz); anti‐tubulin 1:2,500 (Sigma); anti‐IκBα 1:1,000 (Santa Cruz); anti‐β‐actin 1:1,000 (Sigma); anti‐Gapdh 1:10,000 (Sigma); anti‐p27kip1 1:200 (R&D Systems); anti‐mouse 1:10,000 (GE Healthcare); anti‐rabbit 1:10,000 (GE Healthcare).
Villa M., Gialitakis M., Tolaini M., Ahlfors H., Henderson C.J., Wolf C.R., Brink R, & Stockinger B. (2016). Aryl hydrocarbon receptor is required for optimal B‐cell proliferation. The EMBO Journal, 36(1), 116-128.
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2

Protein Detection via Western Analysis and IF

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The primary antibodies for the following proteins were used for Western analysis and immunofluorescence (IF): anti-Nup62 (BD Biosciences #610497, used at 1:2000 for Western), anti-Nup98 (Abcam #45584, used at 1:1000 for Western), anti-Nup153 (Abcam #96462, used at 1:1000 for Western), anti-eIF4G (BD Biosciences #610536, used at 1:1000 for Western), anti-PABP (Cell Signaling Technology #4992, used at 1:1000 for Western), anti-nucleolin (Abcam #22758, used at 1:3000 for Western), anti-hnRNP-C1/C2 (Santa Cruz #32308, used at 1:500 for IF), anti-SC35 (Sigma #4045, used at 1:500 for IF), anti-Sam68 (Santa Cruz #sc333, used at 1:500 for IF), and anti-α/β-tubulin (Cell Signaling Technology #2148, used at 1:1000 for Western). Antibodies to 3Cpro were kindly provided by S. Amineva (Madison, WI, USA; Amineva et al., 2004 (link)) and antibodies to dsRNA were kindly provided by S. Bowden (VIDRL, Melbourne, VIC, Australia).
Walker E.J., Jensen L.M., Croft S, & Ghildyal R. (2015). Variation in the nuclear effects of infection by different human rhinovirus serotypes. Frontiers in Microbiology, 6, 875.
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3

CLIP Assay for Protein-RNA Interactions

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CLIP assays were performed as previously described (Bielli et al., 2014 (link)). In brief, dissociated brain tissue was irradiated three times on ice (100 mJ/cm2). Cell suspension was centrifuged at 4,000 rpm for 3 min, and the pellet was incubated for 10 min on ice in lysis buffer (50-mM Tris, pH 8.0, 100-mM NaCl, 1% NP-40, 1-mM MgCl2, 0.1-mM CaCl2, 0.5-mM Na3VO4, 1-mM DTT, protease inhibitor cocktail [Sigma-Aldrich], and RNase Inhibitor [Promega]). Samples were briefly sonicated and incubated with DNase (RNase-free; Ambion) for 3 min at 37°C and then centrifuged at 15,000 g for 3 min at 4°C. 1 mg of extract was treated with Proteinase K for 30 min at 55°C, and RNA was purified by standard procedure (input) or diluted to 1 ml with lysis buffer and immunoprecipitated using anti-SAM68 (Santa Cruz Biotechnology, Inc.) and anti–hnRNP A1 (Santa Cruz Biotechnology, Inc.) antibodies or IgGs (negative control) in the presence of protein G magnetic Dynabeads (Novox; Life Technologies). 1,000 IU RNase I (Ambion) was added to immunoprecipitates and incubated for 2 h at 4°C under rotation. After stringent washes (Bielli et al., 2014 (link)), an aliquot (10%) was kept as a control of immunoprecipitation, while the rest was treated with 50 µg Proteinase K and incubated for 1 h at 55°C. RNA was then isolated by standard procedures.
Pagliarini V., Pelosi L., Bustamante M.B., Nobili A., Berardinelli M.G., D’Amelio M., Musarò A, & Sette C. (2015). SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy. The Journal of Cell Biology, 211(1), 77-90.
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4

Immunohistochemical Localization of Sam68 and Ki-67

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Tissue slices were dewaxed in xylene and rehydrated by passage through a series of ethanol solutions; subsequently, the tissues were immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven at 121°C for 15 min to retrieve antigen activity. Potential endogenous peroxidase activity was quenched in 3% hydrogen peroxide for 10 min. After rinsing with PBS, tissue sections were blocked with 10% normal goat serum at room temperature for 10 min. Subsequently, they were incubated overnight at 4°C with primary antibodies, anti-Sam68 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-Ki-67 (1:100; Abcam, Cambridge, UK). The negative controls were not incubated with primary antibodies. The next day, tissue sections were rinsed with PBS three times, followed by 30 min incubation at 37°C with polymerized horseradish peroxidase-conjugated anti-rabbit IgG (Boster, Wuhan, China). Antibody binding was visualized using 3,3′-diaminobenzidine solution. The sections were finally counterstained with hematoxylin and were mounted for observation.
Wen H., Li P., Ma H., Zheng J., Yu Y, & Lv G. (2017). High expression of Sam68 in sacral chordomas is associated with worse clinical outcomes. OncoTargets and therapy, 10, 4691-4700.
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5

CLIP Assay for RNA-Protein Interactions

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CLIP assays were performed as previously described (Wang et al., 2009 (link)), with some modification. E13.5 cortices were dissected, freed from meninges and dissociated mechanically. After irradiation with UV light on ice (400 mJ/cm2), samples were incubated with lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1 mM MgCl2; 0.1mMCaCl2; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS, protease inhibitor cocktail, RNase Inhibitor), briefly sonicated and treated with DNase-RNase free for 3 min at 37°C. After centrifugation at 15,000xg for 3 min at 4°C, 500 µg of extract was treated with Proteinase K (PK) for 30 min at 37°C and RNA was purified by standard procedure (input) or diluted to 1 ml with lysis buffer for immunoprecipitation with anti-Sam68 (Santa Cruz, RRID:AB_631869) or control rabbit IgGs (Sigma-Aldrich) as negative control, in presence of protein-G magnetic dynabeads (Life Technologies, United Kingdom) and 10 μl RNase I (1:1000, Ambion, Waltham, MA), for 2 hr at 4°C under rotation. After stringent washes in high salt, dynabeads were resuspended in PK buffer. 10% of each sample was kept as control of immunoprecipitation and the rest was treated with 50 μg PK for 1 hr at 55°C. RNA was then isolated and used for qPCR analysis.
La Rosa P., Bielli P., Compagnucci C., Cesari E., Volpe E., Farioli Vecchioli S, & Sette C. (2016). Sam68 promotes self-renewal and glycolytic metabolism in mouse neural progenitor cells by modulating Aldh1a3 pre-mRNA 3'-end processing. eLife, 5, e20750.
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6

Biotinylated FMDV IRES RNA Binding Assay

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Biotinylated FMDV IRES full length, domain 2, domains 2–4, and domain 4 RNA were transcribed using the Megascript T7 kit (Ambion) with rNTPs supplemented with biotin-11-CTP (Roche). First, 50 μL of streptavidin-M280 magnetic beads (Invitrogen) were pulled-down by a magnet and equilibrated in 1 mL of 1X binding and washing (B&W) buffer consisting of 5 mM Tris–HCl, pH 7.5, 0.5 mM EDTA, and 1 mM NaCl. Following another pulldown and removal of B&W buffer, 5 μg of recombinant Sam68 and 100 μg of tRNA were added to the beads and incubated at 37 ° C for 30 min in a final volume of 200 μL 1X B&W buffer. The beads with potentially bound RNA and protein were pulled-down and washed three times in B&W buffer. Finally, the beads were resuspended in 20 μL of 1X Laemmli sample buffer, heated at 98 ° C for 10 min, and the supernatant collected after centrifugation was loaded onto a 12 % SDS-PAGE gel followed by Western blot analysis probing with anti-Sam68 (Santa Cruz Biotechnology).
Rai D.K., Lawrence P., Kloc A., Schafer E, & Rieder E. (2015). Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections. Virology Journal, 12, 224.
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7

Antibody Validation for Protein Analysis

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Anti-β-actin (R&D Systems, Abingdon, UK #MAB1536), anti-NRF2 (Abcam, Cambridge, UK #62352), anti-GAPDH (Cell Signaling Technology, Cambridge, MA, USA #D16H11), anti-Sam68 (Santa Cruz Biotechnology, Santa Cruz, USA), anti-CHOP (Cell Signaling Technology #1649). All other reagents were obtained from Sigma-Aldrich (St Louis, MO, USA), unless indicated.
Sun Y., Abdul Aziz A., Bowles K, & Rushworth S. (2018). High NRF2 expression controls endoplasmic reticulum stress induced apoptosis in multiple myeloma. Cancer letters, 412.
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8

RNA Immunoprecipitation from Testes

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For RNA immunoprecipitation, testes were dissected from the albuginea membrane, fixed in 4% formaldehyde solution in PBS for 10 min and subsequently quenched by glycine addition (125 mM). Samples were then extracted with RIPA buffer (50 mM Tris pH 7.5; 1% NP-40; 0,5% Na deoxycholate; 0,05% SDS; 150 mM NaCl; 1 mM EDTA; 1 mM DTT; 0,5 mM NaVO 3 ) supplemented with protease inhibitor cocktail (Sigma Aldrich) and 40U/ml RNase inhibitor (Promega), incubated on ice for 30 min, briefly sonicated, and centrifuged for 10 min at 12000 g at 4 C. Lysates were incubated with control or specific primary antibody [anti-Sam68 (Santa Cruz) and Anti-Smith antigen (Y12; Novus Biologicals)] overnight at 4 C under rotation. Next, protein G-Dynabeads were added to lysates and incubated for 2 hours at 4 C under rotation. After rinsing three times with wash buffer [50 mM Tris pH 7.5; 1% NP-40; 1% Na deoxycholate; 0,1% SDS; 500 mM NaCl; 1 mM EDTA; 1 mM DTT; 0,5 mM NaVO 3 ; protease inhibitor cocktail (Sigma Aldrich), RNase inhibitor 40U/ml (Promega)], immunocomplexes were re-suspended in 100 ml TE buffer (10mM Tris, 1mM EDTA, pH 8.0) supplemented with 1% SDS, 10 mM DTT and 40U/ml RNase inhibitor, and incubated for 1 hour at 70 C to reverse crosslinking. After treatment with proteinase K for 1hr at 55 C, RNA was extracted with Trizol, DNase-treated and used for qPCR analysis.
Naro C., Pellegrini L., Jolly A., Farini D., Cesari E., Bielli P., de la Grange P, & Sette C. (2019). Functional Interaction between U1snRNP and Sam68 Insures Proper 3' End Pre-mRNA Processing during Germ Cell Differentiation. Cell reports, 26(11).
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