Flotillin 1 Antikörper (C-Term)

Produktnummer ABIN374222

Produktdetails anti-Flotillin 1 Antikörper

Target: Flotillin 1 (FLOT1)

Bindungsspezifität: C-Term

Reaktivität: Human, Rind (Kuh), Hund, Schwein

Wirt: Ziege

Klonalität: Polyklonal

Konjugat: Dieser Flotillin 1 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))

Spezifität: This antibody detetcs (FLOT1) at C-term.

Kreuzreaktivität (Details): Species reactivity (expected):Dog, Pig, Cow.
Species reactivity (tested):Human.

Aufreinigung: Affinity chromatography

Immunogen: ptide with sequence C-SISQVNHKPLRTA, from the C Terminus of the protein sequence Genename: FLOT1

Anwendungsinformationen

Applikationshinweise: Peptide ELISA: 1/32000. Western blot: 0,3 - 1,0 1 μg/mL. Approx 48 kDa band observed in H460 lysates (predictedMW of 48 kDa according to NP_005794).
Other applications not tested.
Optimal dilutions are dependent on conditions and should be determined by the user.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #101253 (Western Blotting)

by: Internal Disease Clinic, Faculty of Veterinary Medicine, University of Zagreb

No.: #101253

Datum: 03.05.2017

Antigen: Flotillin-1

Validierte Anwendung: Western Blotting

Positivkontrolle: extracellular vesicles from bovine follicular fluid, verified with CD63 antibody

Negativkontrolle: non-enriched fractions

Bewertung: Passed. The flotillin-1 antibody ABIN374222 specifically reveals a band of the expected molecular weight in fractions enriched for exosomes.

Validierungsbilder

Different fractions from an enrichment of extracellular vesicles from bovine follicular fluid were probed with antibodies ABIN374222 and ABIN1440014 against exosome markers flotillin-1 (upper panel) and CD63 (lower panel). See protocol for more information. V0: void volume. 1: fraction 1, some exosomes expected. 2: fraction 2, usually the richest in exosomes. 3: third fraction, lower exosome concentration. 4: fourth fraction, contains no or very few exosomes. FF: follicular fluid.

Protokoll

Primärantikörper: ABIN374222

Sekundärantikörper: anti-goat-HRP

Full Protocol:

• Isolate extracellular vesicles from 0.5ml follicular fluid from cow obtained from abattoir using the qEV size-exclusion chromatography kit (iZON Science, qEVoriginal Size Exclusion Columns, NC0888135):
• • Equilibrate columns with 10ml PBS.
  • Apply the follicular fluid.
  • Retain the first 3ml of the eluate as void volume negative control (V0).
  • Collect the next three 0.5ml fractions (1, 2, 3) which are expected to contain extracellular vesicles.
  • Collect next 0.5ml fraction (4) which is expected to contain proteins but not extracellular vesicles from follicular fluid.
• Concentrate fractions 25x using Microcon filters (Millipore, Ultracel YM-30, MRCF0R30) with a 25kDa cut-off.
• Boil all five fractions and 1µl of untreated follicular fluid as positive control in 1x Laemmli SDS sample buffer for 5min.
• Separate all protein in each fraction, 10µg for fraction 2, the richest in protein on a freshly cast 4% acrylamide stacking gel and 10% acrylamide separation SDS-PAGE gel.
• Transfer proteins onto a nitrocellulose membrane (GE Healthcare,Amersham Protran 0.45µm, 10600002) in an electroblotter tank (Biostep, electroblotting module GV100- EBGRM) Western blotting system using 20% methanol-containing transfer buffer for 2h at 150mA at RT.
• Block the membrane with Odyssey blocking buffer (PBS) (Odyssey, 927-400000) for 1h at RT.
• Incubation with primary goat anti-flotillin 1 antibody (antibodies-online, ABIN374222) diluted 1:500 in blocking buffer ON at 4°C.
• Wash membrane 3x 5min with TBST (TBS, 0.1% Tween-20).
• Incubate with secondary anti-goat-HRP antibody diluted 1:5000 in blocking buffer for 1h at RT.
• Wash membrane 3x 5min with TBST.
• Incubate membrane in Luminol for (Santa Cruz Biotechnology Inc., ImmunoCruz western blotting luminol reagent, SC-2048) 5min at RT.
• Reveal chimiuminescent bands using a chemiluminescence detector (LI-COR, Odyssey Fc, OFC-0966) imaging system.
• Strip membranes usingmild stripping protocol:
• • Incubate membrane with stripping buffer (1.5% (w/v) glycine, 0.0% (w/v) SDS, 1% (v/v) tween 20, pH2.2) shaking for 2x 10min at RT.
  • Wash membrane 2x 10min with PBS.
  • Wash membrane2x 5min in TBS.
  • Block the membrane with blocking buffer.
• Incubate with primary goat anti-CD63 antibody (antibodies-online, ABIN1440014, lot 0047080912) diluted 1:200 in blocking buffer ON at 4°C.
• Repeat the same steps as for the goat anti-flotillin 1 antibody.

Anmerkungen: We observed an albumin band in western blot of some fractions when we used a different secondary (HRP conjugated mouse anti-CD9 antibody) (not shown). Albumin was not observed with ABIN374222.

Handhabung

Konzentration: 0.5 mg/mL

Buffer: Tris saline, pH ~7.3, 0.02 % Sodium Azide, 0.5 % BSA

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handhabung: Avoid repeated freezing and thawing.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Store the antibody undiluted at 2-8 °C for one month or (in aliquots) at -20 °C for longer.

Details zu Flotillin 1

Target: Flotillin 1 (FLOT1)

Andere Bezeichnung: Flotillin-1 / FLOT1 (FLOT1 Produkte)

Hintergrund: Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes: it has been reported to function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton, to associate with caveolae , regulating vesicular trafficking and signal transduction, and to have mitogenic properties. Its subcellular localisation seems to be cell-type-specific. It has been found associated with the cell membranes, nucleus, cytoplasm and different cytoplasmic organelles.Synonyms: REG-2, Reg2, Reggie-2

Gen-ID: 10211

NCBI Accession: NP_005794

UniProt: O75955