Anti-KDEL 抗体 (ab2898)

Three constructs were generated (denoted protein one to three). Proteins 1 and 2 end in SEKDEL as the C-terminal sequence. Protein two is an original construct which ends in GGKDEL. These were expressed and separated on a 12% SDS-PAGE, blotted to PVDF and blocked with 4% milk in TBS/0.1% tween. The replicates were then treated with either a specific antibody for the expressed protein (B), the KDEL antibody - ER Marker (ab2898) or KDEL antibody [10C3] - ER Marker (ab12223). A secondary antibody conjugated to alkaline phosphatase was used to detect the primary. The Coomassie stained gel indicates the location and expression levels of the three proteins. The specific antibody confirms the bands as the proteins of interest. KDEL antibody [10C3] - ER Marker (ab12223) recognizes proteins ending in the SEKDEL sequence and does not recognize the GGKDEL sequence as detected by KDEL antibody - ER Marker (ab2898).

Immunohistochemistry was performed on normal biopsies of deparaffinized Mouse liver tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a rabbit polyclonal antibody recognizing KDEL ab2898 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on normal biopsies of deparaffinized Mouse lymph node tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a rabbit polyclonal antibody recognizing KDEL ab2898 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on normal biopsies of deparaffinized Mouse pancreas tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a rabbit polyclonal antibody recognizing KDEL ab2898 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.