SRSF5 Antikörper (AA 1-272)

Produktnummer ABIN948457

Dieses unabhängig validierte Maus Polyklonal-Antikörper erkennt spezifisch SRSF5 in WB und IF. Er zeigt eine Reaktivität gegenüber Human.

Kurzübersicht für SRSF5 Antikörper (AA 1-272) (ABIN948457)

Target: SRSF5 (serine/arginine-Rich Splicing Factor 5 (SRSF5))

Reaktivität: Human

Wirt: Maus

Klonalität: Polyklonal

Konjugat: Dieser SRSF5 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunofluorescence (IF)

Produktdetails anti-SRSF5 Antikörper

Bindungsspezifität: AA 1-272

Verwendungszweck: Mouse polyclonal antibody raised against a full-length human SFRS5 protein.

Sequenz: MSGCRVFIGR LNPAAREKDV ERFFKGYGRI RDIDLKRGFG FVEFEDPRDA DDAVYELDGK ELCSERVTIE HARARSRGGR GRGRYSDRFS SRRPRNDRRN APPVRTENRL IVENLSSRVS WQDLKDFMRQ AGEVTFADAH RPKLNEGVVE FASYGDLKNA IEKLSGKEIN GRKIKLIEGS KRHSRSRSRS RSRTRSSSRS RSRSRSRSRK SYSRSRSRSR SRSRSKSRSV SRSPVPEKSQ KRGSSSRSKS PASVDRQRSR SRSRSRSVDS GN

Kreuzreaktivität: Human

Produktmerkmale: Antibody reactive against mammalian transfected lysate.

Immunogen: SFRS5 (NP_001034554.1, 1 a.a. ~ 272 a.a) full-length human protein.

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #102043 (Western Blotting)

by: Laboratoire de Biologie et Modélisation de la Cellule, ENS Lyon

No.: #102043

Datum: 27.11.2017

Antigen: SRSF5

Chargennummer: 11159

Validierte Anwendung: Western Blotting

Positivkontrolle: MDA-MB-231 control siRNA, MCF-7 control siRNA

Negativkontrolle: qPCR verified SRSF5 siRNA knockdown in MDA-MB-231 and MCF-7 cells

Bewertung: ABIN948457 specifically reveals SRSF5 in human MDA-MB-231 and MCF-7 cell extracts but also several extraneous protein bands.

Validierungsbilder

A RT-qPCR verified depletion of SRSF5 transcription in MDA-MB-231 (MDA) and MCF-7 cells (MCF) subsequently to transfection with SRSF5 siRNA (siSRSF5) or control siRNA (siGL2). B Western blot analysis using ABIN948457 on cellular extracts with or without siRNA SRSF5 knock-down.

Protokoll

Primärantikörper: ABIN948457

Sekundärantikörper: HRP-conjugated donkey anti-mouse antibody (Bethyl Laboratories, A90-137P)

Full Protocol:

• Grow cells in DMEM (MCF7: Gibco, 10938-025, lot 185560; MDA-MB-231: Gibco, 11880-028, lot 1894718) supplemented with FBS (Gibco, 131608-01, lot 42F6573K) and penicillin-streptomycin (Gibco, 15140-122, lot 1902417), and L-glutamine (Gibco, 25030-024, lot 1895805) 37°C and 5% CO₂.
• Transfect 2.5x10⁶ cells with amount 50nmol of siRNA (eurofins genomics) using Lipofectamine RNAiMAX reagent (Invitrogen, 13778030, lot 1863033) following the manufacturer´s instructions.
• Grow cell for 48h.
• Lyse 5x106 cells in 100µl of cold lysis buffer (50mM Tris-HCl, 400mM NaCl, 5mM EDTA pH8.0, 1% NP40, 0.2% SDS) containing 1x protease inhibitor cocktail (Roche, 05056489001).
• Determine total protein content of the lysates using the Pierce BCA protein assay (ThermoFisher Scientific, 23225, lot sc246917).
• Denature 30µg of total protein for 5min at 95°C in 30µl using NuPAGE Sample Reducing reagent 10x (Invitrogen, np0009, lot 1879834) and the LDS sample buffer (4X) (ThermoFisher Scientific, np0007, lot 1879570) and subsequently separate them on a denaturing gel Bis-Tris 4-12% Protein Gels (ThermoFisher Scientific, np0336box, lot 17061471) in MOPS migration buffer (ThermoFisher Scientific, NP0001, lot 1881690) for 1h at 160V.
• Transfer proteins onto a Trans-Blot Turbo Mini Nitrocellulose membrane (Bio-Rad, 170-4158, lot 64126951) with a Transblot Turbo Transfer System (Bio-Rad, 1704150) for 7min at 1.3A and 25V.
• Block the membrane with TBST (50mM Tris-HCl pH7.4-7.6, 150mM NaCl, 0.1% Tween 20) containing 5% non-fat milk for 1h with slight agitation at RT.
• Incubation with primary mouse anti-human SRSF5 antibody (antibodies-online, ABIN948457, lot 11159) diluted to 1µg/µl in 5ml final volume in TBST + milk ON at 4°C.
• Wash membrane 2x for 10min with TBST.
• Incubation with secondary HRP-conjugated donkey anti-mouse antibody (Bethyl Laboratories, A90-137P) diluted 1:10000 in TBST + milk for 1h at RT.
• Wash membrane 2x for 10min with TBST.
• Reveal protein bands using ECL Prime Western Blotting Detection Reagent (GE healthcare RPN2236, lot 9794767) on a ChemiDoc MP Imaging System (Bio-Rad).

Anmerkungen:

• SRSF5 was depleted to almost 70% in both tested cell lines (Figure A). Quantities are normalized relative to the control siRNA (siGL2) and in comparison with a housekeeping gene (ribosomal 18S RNA, not shown). SRSF5 primers were previously validated on both cell lines. Efficacity of the primers are for the calculation estimated at 2.

• ABIN948457 specifically reveals a protein band with an apparent MW of approximately 45kDa in cell lysates which were transfected with control siRNA (Figure B). Post-translational modifications such as phosphorylation can influence the migration of SRSF5, making it appear bigger than it really is. The band disappears upon knockdown of the SRSF5 expression in cells transfected with an siSRSF5 construct. The antibody does also detect several other proteins at approximately 35-38kDa and 55kDa.

Handhabung

Buffer: In 1x PBS, pH 7.4

Handhabung: Aliquot to avoid repeated freezing and thawing.

Lagerung: -20 °C

Informationen zur Lagerung: Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing.

Details zu SRSF5

Target: SRSF5 (serine/arginine-Rich Splicing Factor 5 (SRSF5))

Andere Bezeichnung: SFRS5

Hintergrund: Full Gene Name: splicing factor, arginine/serine-rich 5
Synonyms: HRS,SRP40

Gen-ID: 6430

NCBI Accession: NM_001039465

Pathways: Ribonucleoprotein Complex Subunit Organization