Anti-Plakophilin 3 antibody [EPR5560]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Plakophilin 3 antibody [EPR5560] (AB109441)

ab109441 at 1/100 dilution staining paraffin-embedded Human kidney tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Plakophilin 3 antibody [EPR5560] (AB109441)

ab109441 at 1/100 dilution staining Human paraffin-embedded breast tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• WB

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Western blot - Anti-Plakophilin 3 antibody [EPR5560] (AB109441)

False colour image of Western blot : Anti-Plakophilin 3 antibody [EPR5560] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109441 was shown to bind specifically to Plakophilin 3. A band was observed at 70 and 80 kDa in wild-type HeLa cell lysates with no signal observed at this size in PKP3 CRISPR-Cas9 edited cell line ab265539 (CRISPR-Cas9 edited cell lysate ab258120). The band observed in the CRISPR-Cas9 edited lysate lane below 70 and 80 kDa is likely to represent a truncated form of Plakophilin 3. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and PKP3 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Plakophilin 3 antibody [EPR5560] (ab109441) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PKP3 CRISPR-Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PKP3 (Plakophilin 3) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-pkp3-plakophilin-3-knockout-hela-cell-lysate-ab258120'>ab258120</a>) at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 87 kDa

Observed band size: 70 kDa,80 kDa,36 kDa

false

• WB

Unknown

Western blot - Anti-Plakophilin 3 antibody [EPR5560] (AB109441)

All lanes:

Western blot - Anti-Plakophilin 3 antibody [EPR5560] (ab109441) at 1/10000 dilution

Lane 1:

Caco-2 cell lysate at 10 µg

Lane 2:

HACAT cell lysate at 10 µg

Predicted band size: 87 kDa

Observed band size: 87 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Plakophilin 3 antibody [EPR5560] (AB109441)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.