Anti-CD66b 抗体 [EPR25354-2] (ab300122)

Anti-CD66b antibody [EPR25354-2] (ab300122) at 1/1000 dilution + Human spleen tissue lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 38 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

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

CD66b is a highly glycosylated protein, the molecular weight observed is consistent with what has been described in the literature (PMID:15147383)
Samples are non-boiled as boiling may cause protein aggregates.
Bis-Tris gel was used for this blot.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labelling CD66b with ab300122 at 1/20000 (0.026 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab300122 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300122) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

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

Immunohistochemical analysis of paraffin-embedded human stomach carcinoma tissue labelling CD66b with ab300122 at 1/20000 (0.026 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Positive staining on neutrophils in human stomach carcinoma (PMID:31329780). The section was incubated with ab300122 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 LeicaDS9800 (BOND™ Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded:

(A) HEK-293T cells transfected with a CD66b expression vector containing a his tag.
(B) HEK-293T cells transfected with a CEACAM1 expression vector containing a his tag.
(C) HEK-293T cells transfected with a CEACAM3 expression vector containing a his tag.
(D) HEK-293T cells transfected with a CEACAM5 expression vector containing a his tag.
(E) HEK-293T cells transfected with a CEACAM6 expression vector containing a his tag.
(F) HEK-293T cells transfected with empty vector containing a his tag. 

All tissues labelling CD66b with ab300122 at 1/20000 (0.027 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). This antibody does not cross-react with CEACAM1, CEACAM3, CEACAM5 and CEACAM6 proteins.
Positive staining on (A) HEK-293T cells transfected with a CEACAM8 expression vector containing a his tag. No staining on HEK-293T cells transfected with a CEACAM1 (B), CEACAM3 (C), CEACAM5 (D), CEACAM6 (E), and empty (F) expression vector containing a his tag. The section was incubated with ab300122 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300150) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

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

Immunohistochemical analysis of paraffin-embedded human stomach tissue labelling CD66b with ab300122 at 1/20000 (0.026 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Positive staining on neutrophils in human stomach (PMID: 32096331). The section was incubated with ab300122 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.  Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody (ab300122) followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

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

CD66b was immunoprecipitated from 0.35 mg Human spleen tissue lysate 10 µg with ab300122 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300122 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 (Input): Human spleen tissue lysate 10 µg

Lane 2: Human spleen tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300122 in Human spleen tissue lysate

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

Exposure time: 7.75 seconds