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BirA (mutated/TurboID)

AS20 4440  | Clonality: Polyclonal |  Host: Rabbit | Reactivity: E. coli BirA - mutated/TurboID overexpressed in various organisms

BirA (mutated/TurboID) in the group Tag Antibodies / GAL4 / GUS / LUC / others at Agrisera AB (Antibodies for research) (AS20 4440)

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Product Information

Immunogen Recombinant mutated BirA protein from E.coli produced using the following plasmid: TurboID-His6_pET21a, (Plasmid #107177). Expression was done in a vector that allowed for the generation of an untagged protein (without HIS6tag).
Host

Rabbit

Clonality Polyclonal
Purity Affinity purified serum, in PBS pH 7.4
Format Lyophilized
Quantity 50 µg
Reconstitution For reconstitution add 50 µl, of sterile water.
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Western blot (WB)
Recommended dilution 1 : 5000 (WB)
Expected | apparent MW 35 kDa

Reactivity

Confirmed reactivity BirA (mutated/TurboID)
Not reactive in No confirmed exceptions from predicted reactivity are currently known

Application examples

Application examples Western blot using anti-TurboID antibodies

1 – 20 µg A. thaliana – Wt Col-0 (Negative control)
2 – 20 µg of A. thaliana expressing POI-TurboID fusion (Independent line 1)
3 – 20 µg of A. thaliana expressing POI-TurboID fusion (Independent line 2)
4 – 20 µg of A. thaliana expressing POI-TurboID fusion (Independent line 3)
5 – 10 ng of purified TurboID (Positive control)

20 µg/well of total protein were extracted from Arabidopsis thaliana leaf material in diluted HENS (25mM HEPES pH 7.7, 1mM EDTA, 2.5 % SDS) and stored at -80°C. Samples were denatured in 1x protein loading dye (0.5% Sodium dodecyl Sulfate, 0.002% Bromophenol Blue, 10% glycerol, and 50 mM Tris-HCl pH6.8) at 95°C for 5 min. Samples were separated on 4-16% gradient SDS-PAGE gel and blotted 1h to a nitrocellulose membrane (pore size of 0.45 um), using a semi-dry transfer. Blot was blocked with 5% milk in TBS-T at 4°C/ON without agitation. Blot was incubated in the primary antibody at a dilution of 1:5000 in 5% milk in TBS-T, at 4°C, ON without agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25 000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 2 min with Agrisera ECLBright. Exposure time was 172 seconds.

Western blot using anti-TurboID antibodies

1 – 1 ng of purified TurboID 2 – 5 ng of purified TurboID 3 – 10 ng of purified TurboID 4 – 25 ng of purified TurboID 5 – 50 ng of purified TurboID 6 – 75 ng of purified TurboID Mark: PageRulerTM Plus Prestained Protein Ladder; ThermoFisher Scientific; MW of TurboID = 35 kDa

1ng, 5 ng, 10 ng, 25 ng, 50 ng, and 75 ng loaded into wells 1, 2, 3, 4, 5, and 6, respectively, of purified TurboID in 1x Phosphate Buffer Saline (PBS) were stored at -80°C. Samples were denatured in 1x protein loading dye (0.5% Sodium dodecyl Sulfate, 0.002% Bromophenol Blue, 10% glycerol, and 50 mM Tris-HCl pH6.8) at 95°C for 5 min. Samples were separated on 4-16% gradient SDS-PAGE gel and blotted 1h to a nitrocellulose membrane (pore size of 0.45 um), using a semi-dry transfer. Blot was blocked with 5% milk in TBS-T at 4°C/ON without agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 in 5% TBS-T, at 4°C, ON without agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed three times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25 000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 2 min with Agrisera ECLBright. Exposure time was 92 seconds.

Courtesy of Eli Gordon and Dr. Patrick Treffon, Elizabeth Vierling Lab Department of Biochemistry and Molecular Biology University of Massachusetts Amherst, USA

Additional information

Related products

Background

Background Tagging a protein with TurboID  allows studying protein interactions in different types of cells and organs and developmental stages. This is a suitable tool for proximity labelling experiments as described in Mair et al. (2019). Proximity labelling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID. Elife . 2019 Sep 19;8:e47864. doi: 10.7554/eLife.47864.

Product citations

Selected references To be added when available, antibody available in January 2021.

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