This is a knockout-validated antibody summary, based on the publication "Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Western blot of UBA5 in control and UBA5-KO H4 neuroglioma cell line (stably expressing Cas9 and GFP-SQSTM1). GAPDH was used as loading control. From [1]. Please note a weak, non-specific band.

Rabbit monoclonal IgG

Company: Abcam

Antibody: UBA5

Catalog number: ab177478

Summary: Rabbit monoclonal IgG against a synthetic peptide within human UBA5 aa 350 to the C-terminus (Cysteine residue). Reacts with human and not with mouse or rat. Suitable for western blot, immunocytochemistry/immunofluorescence, immunohistochemistry (paraffin), flow cytometry. Unsuitable for immunoprecipitation.

Western blot

Control and UBA5-KO H4 neuroglioma cell line (stably expressing Cas9 and GFP-SQSTM1). Cells were lysed in RIPA buffer (Thermo Scientific, Waltham, MA) supplemented with protease inhibitor cocktail (Roche) followed by sonication. Alternatively, protein lysates were prepared in RIPA buffer containing protease inhibitors and 1% SDS followed by purification with QIAshredder columns (Qiagen).

Odyssey blocking buffer for 1 hr or PBS/0.1% Tween-20/5% milk.

Diluted in Odyssey blocking buffer supplemented with 0.1% Tween-20 (Bio-Rad) or in PBS/0.1% Tween-20/5% milk overnight at 4°C.

Anti-rabbit IRDye 800CW (LI-COR) diluted in Odyssey blocking buffer supplemented with 0.1% Tween-20 or anti-rabbit HRP (Thermo Fisher Scientific) diluted in PBS/0.1% Tween-20/5% milk incubated for 1 hr at room temperature.

Odyssey infrared imaging system or SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and autoradiography.

If the antibody described in this summary is a polyclonal antibody, since polyclonal antibodies are of limited quantity, please inquire the supplier whether any current polyclonal antibody with the same catalog number is exactly the same as the one described in this summary. Sometimes, different bleeds or different animals are used, usually with a different lot number. In such cases, the result in this summary may not apply to the new antibody with the same catalog number.