Anti-MUPP1 抗体 [EPR26317-59] (BSA and Azide free) (ab302622)

All lanes : Anti-MUPP1 antibody [EPR26317-59] (ab302621) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell fresh lysate at 20 µg
Lane 2 : Neuro-2a (mouse neuroblastoma neuroblast), whole cell fresh lysate at 20 µg
Lane 3 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell fresh lysate at 20 µg
Lane 4 : SH-SY5Y (human neuroblastoma epithelial cell), whole cell fresh lysate at 20 µg
Lane 5 : C6 (rat glial tumor glial cell), whole cell lysate at 40 µg
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 40 µg
Lane 7 : human cerebellum tissue lysate at 40 µg
Lane 8 : mIMCD3 (mouse inner medullary collecting duct epithelial cell), whole cell lysate at 40 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 219 kDa
Observed band size: 280 kDa why is the actual band size different from the predicted?

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

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

Exposure time:

Lane 1-7: 3 minutes
Lane 8: 26 seconds

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 12403818).

Lysates in lane1-4 were freshly made and used for Western blotting immediately to minimize protein degradation.

The bands beneath the target band are likely to be degraded target fragments.

Samples are non-boiled as boiling may cause protein aggregates.

All lanes : Anti-MUPP1 antibody [EPR26317-59] (ab302621) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate at 40 µg
Lane 2 : Mouse skeletal muscle tissue lysate at 40 µg
Lane 3 : Rat brain tissue lysate at 40 µg
Lane 4 : Rat skeletal muscle tissue lysate at 40 µg
Lane 5 : Mouse testis tissue lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 219 kDa
Observed band size: 280 kDa why is the actual band size different from the predicted?

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

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

Exposure time:

Lane 1-4: 26 seconds
Lane 5: 3 minutes

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 12403818).

Low expression: skeletal muscle (PMID: 12706259)

Lysate in lane 5 was freshly made and used for Western blotting immediately to minimize protein degradation.

The bands beneath the target band are likely to be degraded target fragments.

Samples are non-boiled as boiling may cause protein aggregates.

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse choroid plexus tissue labeling MUPP1 with ab302621 at 1/800 (0.683 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Positive staining in mouse choroid plexus (PMID:30518636, PMID:12706259). The section was incubated with ab302621 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 Leica DS9800 (Bond™ Polymer Refine Detection kit).

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

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat choroid plexus tissue labeling MUPP1 with ab302621 at 1/800 (0.683 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Positive staining in rat choroid plexus (PMID:30518636, PMID:12706259 ). The section was incubated with ab302621 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 Leica DS9800 (Bond™ Polymer Refine Detection kit).

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

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse skeletal muscle tissue labeling MUPP1 with ab302621 at 1/800 (0.683 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Negative control: no staining in mouse skeletal muscle (PMID:12706259). The section was incubated with ab302621 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 Leica DS9800 (Bond™ Polymer Refine Detection kit).

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

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue labeling MUPP1 with ab302621 at 1/800 (0.683 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Negative control: no staining in rat skeletal muscle (PMID:12706259). The section was incubated with ab302621 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 Leica DS9800 (Bond™ Polymer Refine Detection kit).

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

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse choroid plexus tissue labeling MUPP1 with ab302621 at 1/50 (10.92 µg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution (Green). Positive staining on mouse choroid plexus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.<\p>

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat choroid plexus tissue labeling MUPP1 with ab302621 at 1/50 (10.92 µg/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution (Green). Positive staining on rat choroid plexus is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.<\p>

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mIMCD3 (mouse inner medullary collecting duct epithelial cell) cells labeling MUPP1 with AB302621 at 1/50 (10.92 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing membranous and cytoplasmic staining in mIMCD3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A549 (human lung carcinoma epithelial cell) cells labeling MUPP1 with ab302621 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized mIMCD3 (mouse inner medullary collecting duct epithelial cell) cells labeling MUPP1 with ab302621 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab302621, the same antibody clone in a different buffer formulation.

MUPP1 was immunoprecipitated from 0.35 mg C6 (rat glial tumor glial cell), whole cell lysate 10 µg with ab302621 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302621 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C6 (rat glial tumor glial cell), whole cell lysate 10 µg

Lane 2: ab302621 IP in C6 whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab302621 in C6 whole cell lysate

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

The bands beneath the target band are likely to be degraded target fragments.