All lanes : Anti-Granulin antibody [EPR15864] (ab208777) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : GRN knockout HEK293T cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HAP1 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: 64 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab208777 observed at 74 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab208777 Anti-Granulin antibody [EPR15864] was shown to specifically react with Granulin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266738 (knockout cell lysate ab257235) was used. Wild-type and Granulin knockout samples were subjected to SDS-PAGE. ab208777 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Granulin with ab208777 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on trophoblastic cells of Human placenta is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Granulin was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208777 at 1/70 dilution.

Western blot was performed from the immunoprecipitate using ab208777 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate, 10µg (Input).

Lane 2: ab208777 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal -Isotype Control (ab172730) instead of ab208777 in HeLa whole cell lysate.

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

Exposure time: 30 seconds.

All lanes : Anti-Granulin antibody [EPR15864] (ab208777) at 1/2000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : GRN (Granulin) knockout HAP1 whole cell lysate
Lane 3 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 64 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab208777 observed at 64 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab208777 was shown to specifically react with Granulin in wild-type HAP1 cells as signal was lost in GRN (Granulin) knockout cells. Wild-type and GRN (Granulin) knockout samples were subjected to SDS-PAGE. Ab208777 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Lane 1 : Anti-Granulin antibody [EPR15864] (ab208777) at 1/2000 dilution
Lane 2 : Anti-Granulin antibody [EPR15864] (ab208777) at 1/10000 dilution

Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : 293T (Human epithelial cell line from embryonic kidney) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 64 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Granulin antibody [EPR15864] (ab208777) at 1/2000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 64 kDa
Observed band size: 74 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Granulin antibody [EPR15864] (ab208777) at 1/1000 dilution + Human GRN full-length recombinant protein with GST-tag at N-terminal at 0.01 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 64 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Granulin with ab208777 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Human breast cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labeling Granulin with ab208777 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Human gastric cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Granulin with ab208777 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Kupffer cells of Human liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.