All lanes : Anti-TFEB antibody [EPR22941-6] (ab267351) at 1/1000 dilution

Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate
Lane 2 : IM-9 (human multiple myeloma B Lymphoblast whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 53 kDa
Observed band size: 66 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

The molecular weight  observed is consistent with what has been described in the literature (PMID:30519051).

 

TFEB was immunoprecipitated from 0.35 mg IM-9 (human multiple myeloma B Lymphoblast) whole cell lysate with ab267351 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267351 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: IM-9 (human multiple myeloma B Lymphoblast) whole cell lysate 10ug

Lane 2: ab267351 IP in IM-9 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267351 in IM-9 whole cell lysate

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

Exposure time: 2 min.

The weak band around 50kDa is one isoform of TFEB(PMID:28993453).

 

TFEB was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab267351 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267351 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10ug

Lane 2: ab267351 IP in Raji whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267351 in Raji whole cell lysate

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

Exposure time: 15 seconds.

The weak band around 50kDa is one isoform of TFEB(PMID:28993453).

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized IM-9 (human multiple myeloma B Lymphoblast) cells labelling TFEB with ab267351 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). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 100% Methanol-fixed IM-9 (human multiple myeloma B Lymphoblast) cells labelling TFEB with ab267351 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in IM-9 cells. 100% methanol fixation is recommended. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunofluorescent analysis of 100% Methanol-fixed Raji (human Burkitt's lymphoma B lymphocyte) cells labelling TFEB with ab267351 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in Raji cells. 100% methanol fixation is recommended. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is  ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling TFEB with ab267351 at 1/500 dilution (1.042ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human breast cancer (PMID:28017540). The section was incubated with ab256492 for 15 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labeling TFEB with ab267351 at 1/500 dilution (1.042ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human lung cancer (PMID: 26264650) The section was incubated with ab256492 for 15 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling TFEB with ab267351 at 1/500 dilution (1.042ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining human colon (PMID:30519051). The section was incubated with ab256492 for 15 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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