Anti-c-Kit antibody [YR145]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [YR145] (AB32363)

IHC-P image of c-Kit (unpurified ab32363) staining on adult marmoset testis. The sections were fixed in formaldehyde and underwent heat mediated antigen retrieval, using Dako antigen retrieval solution. The sections were then blocked in 5% milk for 30 mins at 25°C. The primary antibody was added at a 1/100 dilution, and then incubated for 18 hours at 4°C. Dapi was used to stain the nuclei (blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [YR145] (AB32363)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue labelling c-Kit with unpurified ab32363.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [YR145] (AB32363)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human seminoma tissue labelling c-Kit with unpurified ab32363.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [YR145] (AB32363)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling c-Kit with unpurified ab32363.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [YR145] (AB32363)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling c-Kit with unpurified ab32363.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [YR145] (AB32363)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human seminoma tissue labelling c-Kit with purified ab32363 at 1/400. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• WB

Lab

Western blot - Anti-c-Kit antibody [YR145] (AB32363)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-c-Kit antibody [YR145] (ab32363) at 1/1000 dilution

All lanes:

Human fetal lung tissue lysate at 10 µg

Secondary

All lanes:

HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/50000 dilution

Predicted band size: 109 kDa

Observed band size: 120-145 kDa

false

• WB

Lab

Western blot - Anti-c-Kit antibody [YR145] (AB32363)

Lanes 1 - 3 : Merged signal (red and green). Green - ab32363 observed at 109 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32363 was shown to recognize 0 in wild-type HAP1 cells as signal was lost at the expected MW in KIT knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and KIT knockout samples were subjected to SDS-PAGE. ab32363 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-c-Kit antibody [YR145] (ab32363) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

KIT knockout HAP1 whole cell lysate at 40 µg

Lane 3:

HUVEC whole cell lysate at 40 µg

Predicted band size: 109 kDa

false

• WB

Lab

Western blot - Anti-c-Kit antibody [YR145] (AB32363)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-c-Kit antibody [YR145] (ab32363) at 1/5000 dilution

All lanes:

Human seminoma tissue lysate at 10 µg

Secondary

All lanes:

HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/50000 dilution

Predicted band size: 109 kDa

Observed band size: 120-145 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-c-Kit antibody [YR145] (AB32363)

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.