(Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel.

Western Blot
Positive WB detected in: HEK293 whole cell lysate(20μg), U-251MG whole cell lysate(20μg), HeLa whole cell lysate(20μg), Raji whole cell lysate(20μg), A549 whole cell lysate(20μg), HepG2 whole cell lysate(20μg)
All lanes: FLOT1 antibody at 1:1000
Secondary
Goat polyclonal to human IgG at 1/40000 dilution
Predicted band size: 47 kDa
Observed band size: 47 kDa
Exposure time：1min

Western Blot
Positive WB detected in: Rat brain tissue lysate(20μg)
All lanes: FLOT1 antibody at 1:1000
Secondary
Goat polyclonal to human IgG at 1/40000 dilution
Predicted band size: 47.4 kDa
Observed band size: 48 kDa
Exposure time: 2min

Immunofluorescence staining of NIH/3T3 cell with CSB-RA008727MA1HU at 1:30 counter-stained with DAPI. The cells were fixed in 4% formaldehyde and blocked in 10% normal Goat Serum. The cells were then incubated with the antibody overnight at 4°C. The secondary antibody was Fluorescein (FITC) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific.

Immunofluorescence staining of Hela cell with CSB-RA008727MA1HU at 1:30 counter-stained with DAPI. The cells were fixed in 4% formaldehyde and blocked in 10% normal Goat Serum. The cells were then incubated with the antibody overnight at 4°C. The secondary antibody was Fluorescein (FITC) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific.

Overlay Peak curve showing A431 cells stained with CSB-RA008727MA1HU (red line) at 1:100. The cells were fixed in 4% formaldehyde and permeated by 0.2% TritonX-100 for 10min. Then 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (1ug/1*10⁶cells) for 45min at 4℃. The secondary antibody used was Fluorescein (FITC) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific at 1:200 dilution for 35min at 4℃.Control antibody (green line) was human IgG1 (1ug/1*10⁶cells) used under the same conditions. Acquisition of >10,000 events was performed.

Western Blot
Positive WB detected in: HepG2 whole cell lysate, K562 whole cell lysate, Hela whole cell lysate, Rat brain tissue, Mouse brain tissue
All lanes: FLOT1 antibody at 3µg/ml
Secondary
Goat polyclonal to rabbit IgG at 1/50000 dilution
Predicted band size: 48, 43 kDa
Observed band size: 48 kDa

IHC image of CSB-PA008727LA01HU diluted at 1:100 and staining in paraffin-embedded human cervical cancer performed on a Leica BondTM system. After dewaxing and hydration, antigen retrieval was mediated by high pressure in a citrate buffer (pH 6.0). Section was blocked with 10% normal goat serum 30min at RT. Then primary antibody (1% BSA) was incubated at 4°C overnight. The primary is detected by a biotinylated secondary antibody and visualized using an HRP conjugated SP system.

IHC image of CSB-PA008727LA01HU diluted at 1:100 and staining in paraffin-embedded human kidney tissue performed on a Leica BondTM system. After dewaxing and hydration, antigen retrieval was mediated by high pressure in a citrate buffer (pH 6.0). Section was blocked with 10% normal goat serum 30min at RT. Then primary antibody (1% BSA) was incubated at 4°C overnight. The primary is detected by a biotinylated secondary antibody and visualized using an HRP conjugated ABC system.

Immunofluorescence staining of Hela cells with CSB-PA008727LA01HU at 1:100, counter-stained with DAPI. The cells were fixed in 4% formaldehyde, permeabilized using 0.2% Triton X-100 and blocked in 10% normal Goat Serum. The cells were then incubated with the antibody overnight at 4°C. The secondary antibody was Alexa Fluor 488-congugated AffiniPure Goat Anti-Rabbit IgG(H+L).

Immunoprecipitating FLOT1 in Hela whole cell lysate
Lane 1: Rabbit control IgG instead of CSB-PA008727LA01HU in Hela whole cell lysate. For western blotting, a HRP-conjugated Protein G antibody was used as the secondary antibody (1/2000)
Lane 2: CSB-PA008727LA01HU (8µg) + Hela whole cell lysate (500µg)
Lane 3: Hela whole cell lysate (10µg)

Immunoprecipitating FLOT1 in K562 whole cell lysate
Lane 1: Rabbit control IgG instead of CSB-PA008727LA01HU in K562 whole cell lysate. For western blotting, a HRP-conjugated Protein G antibody was used as the secondary antibody (1/5000)
Lane 2: CSB-PA008727LA01HU (8µg) + K562 whole cell lysate (500µg)
Lane 3: K562 whole cell lysate (20µg)