Anti-Ly-6K antibody [EPR23021-61]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ly-6K antibody [EPR23021-61] (AB246486)

Immunohistochemical analysis of paraffin-embedded Human heart tissue labeling Ly-6K with ab246486 at 1/2000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Negative control : No staining in human heart (PMID : 18089789). The section was incubated with ab246486 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ly-6K antibody [EPR23021-61] (AB246486)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Ly-6K with ab246486 at 1/2000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in spermatocytes of human testis (PMID : 18089789). The section was incubated with ab246486 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

Unknown

Immunoprecipitation - Anti-Ly-6K antibody [EPR23021-61] (AB246486)

Ly-6K was immunoprecipitated from 0.35 mg Human testis tissue lysate with ab246486 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab246486 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/1000 dilution.

Lane 1 : Human testis tissue lysate 10 ug

Lane 2 : ab246486 IP in Human testis tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab246486 in human testis tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-Ly-6K antibody [EPR23021-61] (ab246486)

Predicted band size: 25 kDa

Observed band size: 24 kDa

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• WB

Lab

Western blot - Anti-Ly-6K antibody [EPR23021-61] (AB246486)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID : 27197181, 18089789).

Negative control : Human heart (PMID : 18089789) Exposure time : 10 seconds

All lanes:

Western blot - Anti-Ly-6K antibody [EPR23021-61] (ab246486) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human heart tissue lysate at 20 µg

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 25 kDa

Observed band size: 24 kDa

false

• WB

Lab

Western blot - Anti-Ly-6K antibody [EPR23021-61] (AB246486)

Lanes 1-2 : Merged signal (red and green). Green - ab246486 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa.

ab246486 Anti-Ly-6K antibody [EPR23021-61] was shown to specifically react with Ly-6K in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265874 (knockout cell lysate ab263248) was used. Wild-type and Ly-6K knockout samples were subjected to SDS-PAGE. ab246486 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Ly-6K antibody [EPR23021-61] (ab246486) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

LY6K knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human LY6K (Ly-6K) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ly6k-ly-6k-knockout-hela-cell-line-ab265874'>ab265874</a>)

Secondary

All lanes:

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 1/10000 dilution

Predicted band size: 25 kDa

Observed band size: 24 kDa

false