Anti-TFEB antibody [EPR22940-151]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22940-151] (AB270604)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji cells labeling TFEB with ab270604 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in Raji cells. 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22940-151] (AB270604)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Raji (Human Burkitt's lymphoma B lymphocyte) cells labeling TFEB with ab270604 at 1/500 (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22940-151] (AB270604)

Flow cytometry overlay histogram showing left Raji positive cells and right negative MCF7 stained with ab270604 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab270604) (1x 106 in 100μl at 0.2μg/ml (1/10350)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22940-151] (AB270604)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling TFEB with ab270604 at 1/500 followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human colon tissue is observed (PMID : 30519051). The section was incubated with ab270604 for 20 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TFEB antibody [EPR22940-151] (AB270604)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized IM-9 cells labeling TFEB with ab270604 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in IM-9 cells. 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TFEB antibody [EPR22940-151] (AB270604)

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling TFEB with ab270604 at 1/500 followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human lung cancer tissue is observed (PMID : 26264650). The section was incubated with ab270604 for 20 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 Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-TFEB antibody [EPR22940-151] (AB270604)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized IM-9 (Human multiple myeloma B Lymphoblast) cells labeling TFEB with ab270604 at 1/500 (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-TFEB antibody [EPR22940-151] (AB270604)

TFEB was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate with ab270604 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270604 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/1000 dilution.

Lane 1 : Raji whole cell lysate.

Lane 2 : ab270604 IP in Raji whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270604 in Raji whole cell lysate.

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

Exposure time : 3 secs.

All lanes:

Immunoprecipitation - Anti-TFEB antibody [EPR22940-151] (ab270604)

Predicted band size: 53 kDa

Observed band size: 66 kDa

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• WB

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Western blot - Anti-TFEB antibody [EPR22940-151] (AB270604)

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

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

Exposure time : 26 secs.

All lanes:

Western blot - Anti-TFEB antibody [EPR22940-151] (ab270604) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 40 µg

Lane 2:

IM-9 (human multiple myeloma B Lymphoblast), whole cell lysate at 40 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

Predicted band size: 53 kDa

Observed band size: 66 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-TFEB antibody [EPR22940-151] (AB270604)

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 ab270604 [EPR22940-151]. 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 [EPR22940-151] (AB270604)

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 ab270604 [EPR22940-151]. 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 [EPR22940-151] (AB270604)

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 ab270604 [EPR22940-151]. 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.