Anti-PCSK9 antibody

• WB

Project

Western blot - Anti-PCSK9 antibody (AB31762)

ab31762 detects a band of ~62 kDa in liver and kidney lysates from mouse and rat. This corresponds to the cleaved form of PCSK9. ab31762 has not detected the 74 kDa proprotein form in the lysates tested.

All lanes:

Western blot - Anti-PCSK9 antibody (ab31762) at 1 µg/mL

Lane 1:

Liver (Mouse) Tissue Lysate at 10 µg

Lane 2:

Kidney (Mouse) Tissue Lysate at 10 µg

Lane 3:

Liver (Rat) Tissue Lysate at 10 µg

Lane 4:

Kidney (Rat) Whole Cell Lysate - normal tissue (<a href='/en-us/products/unavailable/kidney-rat-whole-cell-lysate-normal-tissue-ab29480'>ab29480</a>) at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 74 kDa

Observed band size: 62 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PCSK9 antibody (AB31762)

ICC/IF image of ab31762 stained PC12 cells. The cells were 4% formaldehyde 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 ab31762 at 5μg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PCSK9 antibody (AB31762)

IHC image of ab31762 staining in mouse large intestine formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol B. 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 ab31762, 5µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IP

Unknown

Immunoprecipitation - Anti-PCSK9 antibody (AB31762)

PCSK9 was immunoprecipitated using 0.5mg Mouse Liver tissue, 5μg of Rabbit polyclonal to PCSK9 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, Mouse Liver tissue 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 ab31762.

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

Band : 62kDa; PCSK9

All lanes:

Immunoprecipitation - Anti-PCSK9 antibody (ab31762)

Predicted band size: 74 kDa

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Exposure time: 20min