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Flotillin 1 抗体 (FLOT1) (C-Term)

Details for Product anti-FLOT1 Antibody No. ABIN5552770, 供应商: Log in to see
抗原
  • flot1
  • flot1c
  • flotillin-1
  • flotillin1
  • flot1a
  • reggie-2
  • Rareg
  • reggie2
  • si:dz44o19.1
  • wu:fj45g11
  • flotillin-1
  • Flotillin-1
  • flotillin 1
  • flotillin 1a
  • AaeL_AAEL015235
  • AaeL_AAEL012046
  • CpipJ_CPIJ008623
  • Smp_016200.3
  • Smp_016200.4
  • Smp_016200.1
  • flot1
  • flot1-b
  • flot1-a
  • FLOT1
  • Flot1
  • flot1a
抗原表位
C-Term
18
14
9
9
3
3
3
2
2
1
1
1
1
1
1
1
适用
牛, 犬, 人, 猪
86
34
20
11
10
9
7
6
4
4
3
2
2
1
1
1
宿主
山羊
61
21
6
克隆类型
多克隆
标记
This Flotillin 1 antibody is un-conjugated
6
4
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
应用范围
Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Western Blotting (WB)
85
38
10
7
5
5
2
1
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'独立验证'标志
抗原 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 ABIN5552770 specifically reveals a band of the expected molecular weight in fractions enriched for exosomes.
Primary Antibody ABIN5552770
Secondary Antibody anti-goat-HRP
实验流程
  • 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, ABIN5552770) 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 ABIN5552770.
Validation Images
Western Blotting validation image for anti-Flotillin 1 (FLOT1) (C-Term) antibody (ABIN5552770) Different fractions from an enrichment of extracellular vesicles from bovine follicul...
免疫原 ptide with sequence C-SISQVNHKPLRTA, from the C Terminus of the protein sequence Genename: FLOT1
特异性 This antibody detetcs (FLOT1) at C-term.
交叉反应 (详细) Species reactivity (expected):Dog, Pig, Cow.
Species reactivity (tested):Human.
纯化方法 Affinity chromatography
别名 Flotillin-1 / FLOT1 (FLOT1 Antibody 摘要)
背景 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
基因ID 10211
NCBI登录号 NP_005794
UniProt O75955
应用备注 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.
限制 仅限研究用
浓度 0.5 mg/mL
缓冲液 Tris saline, pH ~7.3, 0.02 % Sodium Azide, 0.5 % BSA
储存液 Sodium azide
注意事项 This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
注意事项 Avoid repeated freezing and thawing.
储存条件 4 °C/-20 °C
储存方法 Store the antibody undiluted at 2-8 °C for one month or (in aliquots) at -20 °C for longer.