PFKM Protein, Human (HEK293, His)

PFKM protein, a key regulator in cellular metabolism, catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate, initiating glycolysis. This fundamental enzymatic step controls glucose entry into glycolysis, influencing downstream energy production and cellular metabolism. PFKM's pivotal role highlights its significance in orchestrating metabolic flux and maintaining cellular energy balance. PFKM Protein, Human (HEK293, His) is the recombinant human-derived PFKM protein, expressed by HEK293 , with C-6*His labeled tag.

The PFKM protein assumes a pivotal role in cellular metabolism by catalyzing the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate using ATP as a substrate. This enzymatic activity marks the initial and committing step of glycolysis, a fundamental metabolic pathway central to energy production. By facilitating the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate, PFKM plays a key role in regulating the entry of glucose into the glycolytic pathway, thereby influencing downstream energy production and cellular metabolic processes. The significance of PFKM in catalyzing this essential reaction underscores its importance in orchestrating the metabolic flux and maintaining cellular energy balance.

Measured by its ability to phosphorylate fructose-6-phosphate.The specific activity is 502.29 pmol/min/μg, as measured under the described conditions.

Materials
Assay buffer (10x): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl₂, 100 mM CaCl₂, pH 7.0
PFKM Protein, Human (HEK293, His) (HY-P71090)
Substrate: ATP, Fructose-6-Phosphate (HY-137834)
Standard: ATP

Procedure
1. Dilute the 10x assay buffer with deionized water to prepare 1xassay buffer.
2. Reaction Mixture: Prepare a reaction mixture containing 0.05 mM ATP and 2.5 mM Fructose-6-Phosphate in 1x assay buffer.
3. Dilute Human PFKM to 10 μg/mL in 1x assay buffer.
4. Standard Curve Preparation: Dilute ATP to 0, 0.3, 0.6, 1.2, 2.5, 5, 10, 20, 40, 50 μM in 1x assay buffer. Add 50 μL of each concentration to the wells as standard wells.
5. Experimental Group: Mix 20 μL of 100 μg/mL PFKM protein with 30 μL of reaction mixture.
Blank Group: Mix 20 μL of 1X Assay Buffer with 30 μL of reaction mixture.
6. Add 50 μL of Kinase-Lumi^TM Enhanced Chemiluminescent Kinase Detection Reagent to Reagent to the standard wells, experimental wells, and blank wells. Mix thoroughly.
7. Incubate at 25oC for 10 minutes, then perform chemiluminescence detection using a multimode microplate reader.
8. Calculate specific activity:

Human

HEK293

C-6*His

P08237-1 (T2-V780)

5213  [NCBI]

Full Length of Isoform-1 Mature Protein

THEEHHAAKTLGIGKAIAVLTSGGDAQGMNAAVRAVVRVGIFTGARVFFVHEGYQGLVDGGDHIKEATWESVSMMLQLGGTVIGSARCKDFREREGRLRAAYNLVKRGITNLCVIGGDGSLTGADTFRSEWSDLLSDLQKAGKITDEEATKSSYLNIVGLVGSIDNDFCGTDMTIGTDSALHRIMEIVDAITTTAQSHQRTFVLEVMGRHCGYLALVTSLSCGADWVFIPECPPDDDWEEHLCRRLSETRTRGSRLNIIIVAEGAIDKNGKPITSEDIKNLVVKRLGYDTRVTVLGHVQRGGTPSAFDRILGSRMGVEAVMALLEGTPDTPACVVSLSGNQAVRLPLMECVQVTKDVTKAMDEKKFDEALKLRGRSFMNNWEVYKLLAHVRPPVSKSGSHTVAVMNVGAPAAGMNAAVRSTVRIGLIQGNRVLVVHDGFEGLAKGQIEEAGWSYVGGWTGQGGSKLGTKRTLPKKSFEQISANITKFNIQGLVIIGGFEAYTGGLELMEGRKQFDELCIPFVVIPATVSNNVPGSDFSVGADTALNTICTTCDRIKQSAAGTKRRVFIIETMGGYCGYLATMAGLAAGADAAYIFEEPFTIRDLQANVEHLVQKMKTTVKRGLVLRNEKCNENYTTDFIFNLYSEEGKGIFDSRKNVLGHMQQGGSPTPFDRNFATKMGAKAMNWMSGKIKESYRNGRIFANTPDSGCVLGMRKRALVFQPVAELKDQTDFEHRIPKEQWWLKLRPILKILAKYEIDLDTSDHAHLEHITRKRSGEAAV

Approximately 90.0-93.0 kDa

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Solution

Supplied as a 0.2 μm filtered solution of 20 mM PB, 150 mM NaCl, 5 mM EDTA, 5% Trehalose, pH 6.9.

<1 EU/μg, determined by LAL method.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.