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.

All lanes : 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
Lane 2 : LY6K knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 19 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

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

Lane 1 : Human testis tissue lysate
Lane 2 : Human heart tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 19 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

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

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.

 

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.