Anti-YKL-40/CHI3L1 antibody [EPR19078-157]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (AB255297)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanolpermeabilized THP-1 (human monocytic leukemia cell line) cells labelling YKL-40/CHI3L1 with ab255297 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (AB255297)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling YKL-40/CHI3L1 with ab255297 at 1/250 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on macrophages in human tonsil germinal center is observed. The section was incubated with ab255297 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (AB255297)

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling YKL-40/CHI3L1 with ab255297 at 1/250 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on neutrophils of human spleen is observed.

The section was incubated with ab255297 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

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

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

• WB

Lab

Western blot - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (AB255297)

False colour image of Western blot : Anti-YKL-40/CHI3L1 antibody [EPR19078-157] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab255297 was shown to bind specifically to YKL-40/CHI3L1. A band was observed at 37/40 kDa in wild-type THP-1 cell lysates with no signal observed at this size in CHI3L1 knockout cell line ab280038 (knockout cell lysate ab280097). To generate this image, wild-type and CHI3L1 knockout THP-1 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-YKL-40/CHI3L1 antibody [EPR19078-157] (ab255297) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human CHI3L1 knockout THP-1 cell lysate (<a href='/en-us/products/cell-lysates/human-chi3l1-knockout-thp-1-cell-lysate-ab280097'>ab280097</a>) at 20 µg

Lane 3:

U-87 MG 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: 43 kDa

Observed band size: 37 kDa,40 kDa,55 kDa

false

• WB

Lab

Western blot - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (AB255297)

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

The expression profile observed in 293T and Jurkat are consistent with the literature (PMID : 19767768，PMID : 16472595).

All lanes:

Western blot - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (ab255297) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 20 µg

Lane 2:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 3:

U-87 MG ( Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

MG-63 (Human osteosarcoma fibroblast) whole cell lysate at 20 µg

Lane 5:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 7:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 27 kDa,42 kDa

false

Exposure time: 60s

• WB

Unknown

Western blot - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (AB255297)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 3 minutes

The molecular weight observed is consistent with what has been described in the literature (PMID 19414556).

All lanes:

Western blot - Anti-YKL-40/CHI3L1 antibody [EPR19078-157] (ab255297) at 1/1000 dilution

Lane 1:

Human spleen lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia cell line) whole cell lysate at 20 µg

Secondary

Lane 1:

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

Lane 2:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 42 kDa

false