Flotillin 1 antibody (C-Term)

Catalog No. ABIN374222

The Goat Polyclonal anti-Flotillin 1 antibody has been validated for WB and IHC (p) and has been independently validated. It is suitable to detect Flotillin 1 in samples from Human, Cow, Dog and Pig.

Quick Overview for Flotillin 1 antibody (C-Term) (ABIN374222)

Target: Flotillin 1 (FLOT1)

Reactivity: Human, Cow, Dog, Pig

Host: Goat

Clonality: Polyclonal

Conjugate: This Flotillin 1 antibody is un-conjugated

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

Product Details anti-Flotillin 1 Antibody

Binding Specificity: C-Term

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

Cross-Reactivity (Details): Species reactivity (expected):Dog, Pig, Cow.
Species reactivity (tested):Human.

Purification: Affinity chromatography

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

Application Details

Application Notes: 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.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101253 (Western Blotting)

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

No.: #101253

Date: 05/03/2017

Antigen: Flotillin-1

Method validated: Western Blotting

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

Negative Control: non-enriched fractions

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

Validation Images

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.

Full Methods

Primary Antibody: ABIN374222

Secondary Antibody: 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.

Experimental Notes: 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.

Handling

Concentration: 0.5 mg/mL

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

Preservative: Sodium azide

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

Handling Advice: Avoid repeated freezing and thawing.

Storage: 4 °C/-20 °C

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

Target Details for Flotillin 1

Target: Flotillin 1 (FLOT1)

Alternative Name: Flotillin-1 / FLOT1

Background: 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

Gene ID: 10211

NCBI Accession: NP_005794

UniProt: O75955