BirA Protein, E. coli (His-MBP)

BirA Protein, E. coli (His-MBP) is the recombinant E. coli-derived BirA protein, expressed by E. coli , with N-His, N-MBP labeled tag.

WBP2 protein functions as a transcriptional coactivator for estrogen and progesterone receptors (ESR1 and PGR) upon hormone activation, binding to ESR1-responsive promoters in the presence of estrogen. It is essential for the coactivation function of YAP1 on PGR activity and synergizes with WBP2 in enhancing PGR activity. Additionally, WBP2 modulates the expression of post-synaptic scaffolding proteins through the regulation of ESR1, ESR2, and PGR. WBP2 interacts with the WW domain of YAP1, WWP1, and WWP2, and also forms interactions with NEDD4. Moreover, it associates with ESR1 and UBE3A, contributing to its role as a transcriptional coactivator in hormone-responsive pathways.

Measured by its ability to generate pyrophosphate from the biotinylation reaction. The pyrophosphate is subsequently hydrolyzed using Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (HY-P79310) that incubated at room temperature for 15 min. The specific activity is >100 pmol/min/μg.

Materials
Assay buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
BirA Protein, E. coli (His-MBP) (HY-P75475)
PPA1 Protein, S. cerevisiae (His) (HY-P79310)
OX40/TNFRSF4 Protein, Human (HEK293, Fc) (HY-P70813)
Magnesium chloride: Prepare 1 M MgCl₂ in deionized water
Biotin: Prepare 10 mM biotin in DMSO
Adenosine triphosphate (ATP): Prepare 400 mM ATP in deionized water
Malachite green phosphate detection kit

Procedure
1. Preparation of phosphate standard solution: Dilute 5 mM phosphate standard stock solution to a 50 μM phosphate working solution. Take 10 μL of 5 mM phosphate standard stock solution and add 990 μL of deionized water to prepare the standard dilution solution, yielding 1 mL of 50 μM phosphate working solution.
2. Standard Curve Preparation: Prepare 200 μL aliquots of phosphate standards at 0, 1, 2.5, 5, 10, 20, 30, 40, and 50 μM according to the kit instructions. Include a curve blank containing 200 μL assay buffer.
3. Prepare 1 mL reaction mixture containing 2 mM ATP, 20 mM MgCl₂, 800 μM Biotin, 280 μg/mL Human OX40, and 8 μg/mL Yeast PPA1 using assay buffer.
4. Dilute E. coli BirA to 40 μg/mL using assay buffer.
5. Add 100 μL of 40 μg/mL E. coli BirA to a 96-well transparent plate. Include 100 μL assay buffer as a blank control. Perform triplicate wells for each concentration.
6. Add 100 μL of reaction mixture to wells without the standard curve and incubate for 15 min.
7. Color development reagent preparation: Based on sample volume, prepare the color development reagent by mixing 1000 μL Malachite Green Reagent A and 500 μL Malachite Green Reagent B in a 2:1 ratio.
8. Phosphate concentration detection
a. Pipette 200 μL of each phosphate standard and sample concentration sequentially into the sample wells of a 96-well plate, including a curve blank containing 200 μL assay buffer.
b. Add 70 μL of color developer to each well. Gently mix by pipetting and incubate at room temperature for 10 min.
c. Calculate phosphate concentration based on the standard curve and sample dilution factor. Read OD at 630 nm in endpoint mode.
10. Calculate Specific Activity:

E.coli

E. coli

N-6*His;N-MBP

P06709 (M1-K321)

948469  [NCBI]

Full Length

MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK

Approximately 64-77 kDa

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder

1.Lyophilized from a 0.22 μm filtered solution of 50 mM Tris, 100 mM NaCl, 10% glycerol, pH 8.0, 5% trehalose, 5% mannitol, 0.01% Tween 80.
2.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4, 8% trehalose, 8% mannitol, 0.02% Tween 80.
Please refer to the lot-specific COA for specific buffer information.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.