All lanes : Anti-Tmem27 antibody [EPR17748] (ab200664) at 1/1000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal brain lysate
Lane 3 : Human fetal heart lysate
Lane 4 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

The observed MW is consistent with what has been described in the literature PMID:16330323.

Please note lanes 2-4 are negative controls for Tmem27.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK293 (Human embryonic kidney) cells labeling Tmem27 with ab200664 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Cytoplasm staining on HEK293 cell line is observed.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab200664 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized BxPC-3 (Human pancreas adenocarcinoma cells) cells labeling Tmem27 with ab200664 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Cytoplasm staining on BxPC-3 cell line is observed.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab200664 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

All lanes : Anti-Tmem27 antibody [EPR17748] (ab200664) at 1/1000 dilution

Lane 1 : Human pancreas lysate
Lane 2 : Human fetal brain lysate
Lane 3 : Human fetal heart lysate
Lane 4 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

The observed MW is consistent with what has been described in the literature PMID:16330323.

Please note lanes 2-4 are negative controls for Tmem27.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Tmem27 antibody [EPR17748] (ab200664) at 1/1000 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse brain lysate
Lane 3 : Mouse heart lysate
Lane 4 : Mouse spleen lysate
Lane 5 : Rat kidney lysate
Lane 6 : Rat brain lysate
Lane 7 : Rat heart lysate
Lane 8 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

The observed MW is consistent with what has been described in the literature PMID:16330323.

Please note lanes 2-4 and 6-8 are negative controls for Tmem27.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Tmem27 with ab200664 at 1/10000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Luminal surface and cytoplasm staining on Human kidney tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling Tmem27 with ab200664 at 1/10000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Luminal surface and cytoplasm staining on rat kidney tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Tmem27 with ab200664 at 1/10000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

No staining on Human cerebral cortex tissue is observed (Negative control for Tmem27).

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 2% paraformaldehyde-fixed BxPC-3 (Human pancreas adenocarcinoma cells) cells labeling Tmem27 with ab200664 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.