Anti-TFEB antibody [EPR22941-6]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (AB267351)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (AB267351)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22941-6] (AB267351)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (AB267351)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22941-6] (AB267351)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22941-6] (AB267351)

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.

• IP

Unknown

Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (AB267351)

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).

All lanes:

Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)

Predicted band size: 53 kDa

Observed band size: 66 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (AB267351)

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).

All lanes:

Immunoprecipitation - Anti-TFEB antibody [EPR22941-6] (ab267351)

Predicted band size: 53 kDa

Observed band size: 66 kDa

false

• WB

Supplier Data

Western blot - Anti-TFEB antibody [EPR22941-6] (AB267351)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : MCF7. The lysates were freshly made and used for Western blotting immediately to minimize protein degradation. In Western blot, anti-TFEB antibody (ab267351) staining at 1/1000 dilution.Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 81 seconds

All lanes:

Western blot - Anti-TFEB (phospho S211) antibody [EPR27960-64] (<a href='/en-us/products/primary-antibodies/tfeb-phospho-s211-antibody-epr27960-64-ab310331'>ab310331</a>) at 1/1000 dilution

Lane 1:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 70 kDa

false

Exposure time: 81s

• WB

Unknown

Western blot - Anti-TFEB antibody [EPR22941-6] (AB267351)

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).

All lanes:

Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 10 µg

Lane 2:

IM-9 (human multiple myeloma B Lymphoblast whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 53 kDa

Observed band size: 66 kDa

false

• WB

Lab

Western blot - Anti-TFEB antibody [EPR22941-6] (AB267351)

False colour image of Western blot : Anti-TFEB antibody [EPR22941-6] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab267351 was shown to bind specifically to TFEB. A band was observed at 60 kDa in wild-type HepG2 cell lysates with no signal observed at this size in TFEB knockout cell line ab277871 (knockout cell lysate ab284220). To generate this image, wild-type and TFEB knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-TFEB antibody [EPR22941-6] (ab267351) at 1/1000 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

TFEB knockout HepG2 cell lysate at 20 µg

Lane 2:

Western blot - Human TFEB knockout Hep G2 cell line (<a href='/en-us/products/cell-lines/human-tfeb-knockout-hep-g2-cell-line-ab277871'>ab277871</a>)

Lane 3:

Raji cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Predicted band size: 53 kDa

Observed band size: 60 kDa

false

• WB

Supplier Data

Western blot - Anti-TFEB antibody [EPR22941-6] (AB267351)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression of TFEB (phospho S211) is downregulated in response to Torin 1 treatment (PMID : 28055300). In Western blot, anti--TFEB antibody (ab267351) staining at 1/1000 dilution.Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 81 seconds

All lanes:

Western blot - Anti-TFEB (phospho S211) antibody [EPR27960-64] (<a href='/en-us/products/primary-antibodies/tfeb-phospho-s211-antibody-epr27960-64-ab310331'>ab310331</a>) at 1/1000 dilution

Lane 1:

Untreated Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Raji treated with 250 nM Torin 1 for 5 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 70 kDa

false

Exposure time: 81s

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-TFEB antibody [EPR22941-6] (AB267351)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 Raji (human Burkitt's lymphoma B lymphocyte) cells and 5 µg of ab267351 [EPR22941-6]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-TFEB antibody [EPR22941-6] (AB267351)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 Raji (human Burkitt's lymphoma B lymphocyte) cells and 5 µg of ab267351 [EPR22941-6]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-TFEB antibody [EPR22941-6] (AB267351)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 Raji (human Burkitt's lymphoma B lymphocyte) cells and 5 µg of ab267351 [EPR22941-6]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.