Anti-ALK-1抗体

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ALK-1 antibody (AB68703)

IHC image of ALK-1 staining in normal human liver formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab68703, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ALK-1 antibody (AB68703)

ICC/IF image of ab68703 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab68703, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HepG2 and MCF7 cells at 5µg/ml.

• IP

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Immunoprecipitation - Anti-ALK-1 antibody (AB68703)

ALK-1 was immunoprecipitated using 0.5mg K562 whole cell extract, 5μg of Rabbit polyclonal to ALK-1 and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, K562 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab68703.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 62kDa; ALK-1, non specific - as present in control (lane 2); We are confident this was due to slight lane contamination and the band seen in the IP lane is our target of interest.

All lanes:

Immunoprecipitation - Anti-ALK-1 antibody (ab68703)

Predicted band size: 56 kDa

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Exposure time: 4min

• WB

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Western blot - Anti-ALK-1 antibody (AB68703)

ALK-1 contains a number of potential glycosylation and phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

All lanes:

Western blot - Anti-ALK-1 antibody (ab68703) at 1 µg/mL

Lane 1:

K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate at 10 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 56 kDa

Observed band size: 62 kDa

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