Anti-PTBP1 antibody [EPR9048(B)]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

Intracellular Flow Cytometry analysis of A549 (Human lung carcinoma epithelial cell) cells labeling PTBP1 (red) with purified ab133734 at a 1/2000 dilution (1ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

Immunohistochemical analysis of PTBP1 in paraffin embedded Human breast carcinoma tissue stained with ab133734 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

Immunofluorescent staining of PTBP1 in A549 cells, using ab133734 at a 1/250 dilution.

• WB

Lab

Western blot - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

Lanes 1- 2 : Merged signal (red and green). Green - ab133734 observed at 57 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab133734 was shown to react with PTBP1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265155 (knockout cell lysate ab257614) was used. Wild-type HeLa and PTBP1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab133734 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PTBP1 antibody [EPR9048(B)] (ab133734) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PTBP1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PTBP1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ptbp1-knockout-hela-cell-line-ab265155'>ab265155</a>)

Predicted band size: 57 kDa

Observed band size: 57 kDa

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• WB

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Western blot - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

All lanes:

Western blot - Anti-PTBP1 antibody [EPR9048(B)] (ab133734) at 1/10000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

Daudi cell lysate at 10 µg

Lane 4:

A549 cell lysate at 10 µg

Lane 5:

BxPC3 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 57 kDa

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• WB

Supplier Data

Western blot - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

Lanes 1 - 2 : Merged signal (red and green). Green - ab133734 observed at 57 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab133734 was shown to recognize PTBP1 in wild-type HAP1 cells as signal was lost at the expected MW in PTBP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PTBP1 knockout samples were subjected to SDS-PAGE. ab133734 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PTBP1 antibody [EPR9048(B)] (ab133734) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

PTBP1 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 57 kDa

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• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-PTBP1 antibody [EPR9048(B)] (AB133734)

Immunocytochemistry-immunofluorescence using Anti-PTBP1 antibody [EPR9048(B)], ab133734. Publication image from Makeyev, E. V. et al., 2022, Mol Cell, 34741808. Legend direct from paper.

Hybridization-proximity labeling technology for genetically unperturbed samples(A) Top : recombinant HyPro enzyme containing the APEX2 and digoxigenin-binding domains connected by a flexible linker. Bottom : HyPro-labeling approach to identify proteins and RNAs colocalizing with an RNA target of interest.(B) SDS-PAGE analysis of purified HyPro protein peak eluted from a size exclusion chromatography column.(C) Peroxidase activity assay of purified HyPro enzyme.(D) Spot assay showing that HyPro enzyme can bind digoxigenin-labeled DNA while retaining the peroxidase activity.(E) Workflows developed to identify proteins (HyPro-MS) and RNAs (HyPro-seq) colocalized with a transcript of interest and to validate specificity of digoxigenin-labeled probes (HyPro-FISH).(F–H) HyPro-FISH analyses of (F) the 45S pre-rRNA, (G) NEAT1, and (H) the (UC)n-repeated part of PNCTR combined with immunofluorescence detection of nucleolar (No) marker FBL, paraspeckle (Ps) marker SFPQ, and PNC marker PTBP1. As expected, FBL but not 45S is detectable in Cajal bodies (Cb). Image acquisition settings were adjusted according to RNA bait abundance. All images in (F)–(H) are maximum-intensity z stacks. Scale bars, 5 µm.See also Figure S1.