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E3 ligase conjugation buffer

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E3 Ligase Conjugation Buffer is a buffer solution designed for use in enzymatic reactions involving E3 ligase enzymes. The buffer is formulated to maintain optimal conditions for E3 ligase activity, supporting the conjugation of ubiquitin or ubiquitin-like molecules to target proteins.

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Lab products found in correlation

Ubiquitin, by R&D Systems (2 mentions) Anti strep hrp conjugate, by Thermo Fisher Scientific (2 mentions) Mg atp, by R&D Systems (2 mentions) Odyssey clx, by LI COR (2 mentions) Ube2d2, by R&D Systems (2 mentions) Amersham imager 600, by GE Healthcare (2 mentions) Anti flag m2 antibody, by Merck Group (2 mentions) Amersham ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)

2 protocols using e3 ligase conjugation buffer

1

In vitro Ubiquitylation Assay

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0.5 μM UBE2D2 (R&D Systems) was charged with ubiquitin for 30 min by the addition of 1X E3 Ligase Conjugation Buffer (R&D Systems), 0.2 μM UBE1 (R&D Systems), 50 μM ubiquitin (R&D Systems), and 10 mM MgATP (R&D systems). In vitro ubiquitylation reactions were initiated by addition of 2 μM Strep-HAPSTR1 variant or FLAG-SCNM1 and 0.2 μM FLAG-HUWE1 or Strep-HUWE1, respectively. Reactions were allowed to proceed at the indicated temperature for 20 min, quenched by addition of reducing SDS dye and analyzed via immunoblot with a 1:16,000 dilution of an anti-Strep HRP conjugate (Fisher Scientific) or with a 1:1000 dilution of an anti-FLAG M2 antibody (Sigma Aldrich, F3165). Blots were imaged on an Amersham Imager 600 using Amersham ECL Prime Western Blotting Detection Reagent (GE Life Sciences) or on an LI-COR Odyssey CLx detecting an anti-mouse secondary antibody (LI-COR, 926–68070).
Monda J.K., Ge X., Hunkeler M., Donovan K.A., Ma M.W., Jin C.Y., Leonard M., Fischer E.S, & Bennett E.J. (2023). HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways. Cell reports, 42(5), 112496.
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2

In vitro Ubiquitylation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
0.5 μM UBE2D2 (R&D Systems) was charged with ubiquitin for 30 min by the addition of 1X E3 Ligase Conjugation Buffer (R&D Systems), 0.2 μM UBE1 (R&D Systems), 50 μM ubiquitin (R&D Systems), and 10 mM MgATP (R&D systems). In vitro ubiquitylation reactions were initiated by addition of 2 μM Strep-HAPSTR1 variant or FLAG-SCNM1 and 0.2 μM FLAG-HUWE1 or Strep-HUWE1, respectively. Reactions were allowed to proceed at the indicated temperature for 20 min, quenched by addition of reducing SDS dye and analyzed via immunoblot with a 1:16,000 dilution of an anti-Strep HRP conjugate (Fisher Scientific) or with a 1:1000 dilution of an anti-FLAG M2 antibody (Sigma Aldrich, F3165). Blots were imaged on an Amersham Imager 600 using Amersham ECL Prime Western Blotting Detection Reagent (GE Life Sciences) or on an LI-COR Odyssey CLx detecting an anti-mouse secondary antibody (LI-COR, 926–68070).
Monda J.K., Ge X., Hunkeler M., Donovan K.A., Ma M.W., Jin C.Y., Leonard M., Fischer E.S, & Bennett E.J. (2023). HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways. Cell reports, 42(5), 112496.
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