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CS0340

Sigma-Aldrich

Dihydrofolate Reductase Assay Kit

1 kit sufficient for 50-100 tests

Synonym(s):

DHFR Assay Kit

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About This Item

UNSPSC Code:
12161503
NACRES:
NA.84
Pricing and availability is not currently available.

Quality Level

200

usage

 kit sufficient for 50-100 tests

shipped in

dry ice

storage temp.

−20°C

Gene Information

human ... DHFR(1719)

Application

The kit is used for the detection of DHFR activity and screening of DHFR inhibitors. The assay is based on the ability of DHFR to catalyze the reversible NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid. The reaction progress is followed by monitoring the decrease in absorbance at 340 nm.
Dihydrofolic acid+NADPH+H+ ↔ Tetrahydrofolic acid+NADP+

Biochem/physiol Actions

DHFR (dihydrofolate reductase) is an enzyme that catalyzes a reaction essential for the biosynthesis of nucleotidic bases of DNA. Blocking the enzyme causes cell death as a result of DNA synthesis inhibition. DHFR is an excellent target for anti-tumor drugs.

Features and Benefits

  • Quick and simple method.
  • The kit contains all the reagents required for a colorimetric assay of DHFR activity in cell lysates, tissue homogenates, or column fractions of purified enzyme.
  • The kit includes a purified enzyme for use as a positive control and screening of DHFR inhibitors.
  • The kit includes methotrexate (MTX), a prokaryotic and eukaryotic DHFR specific inhibitor, which exhibits anti-tumor activities.
  • The kit was tested on A431, NIH-3T3, and CHO cell lines, rat liver, kidney, brain, and skeletal muscle tissue extracts, and recombinant DHFR.

Kit Components Only

Product No.
Description
  • Assay Buffer 10x for DHFR 30 mL
  • Dihydrofolate Reductase (DHFR) human .1 U
  • Dihydrofolic acid (DHFR substrate) 3 x 10
  • Amethopterin (+)(methotrexate, MTX)
    (DHFR inhibitor) 2 x 10
  • NADPH (β-Nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt) 25 mg

Pictograms

Skull and crossbonesHealth hazard
GHS06,GHS08

Signal Word

Danger

Hazard Classifications

Acute Tox. 3 Oral - Eye Irrit. 2 - Repr. 1B - Skin Irrit. 2 - STOT SE 3

Target Organs

Respiratory system

Storage Class Code

6.1C - Combustible acute toxic Cat.3 / toxic compounds or compounds which causing chronic effects

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

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Certificates of Analysis (COA)

Lot/Batch Number
0000381587
0000381587
0000306369
0000191841
0000260052

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Marta Jorba et al.
Antibiotics (Basel, Switzerland), 10(6) (2021-07-03)
This work reports a detailed characterization of the antimicrobial profile of two trimethoprim-like molecules (compounds 1a and 1b) identified in previous studies. Both molecules displayed remarkable antimicrobial activity, particularly when combined with sulfamethoxazole. In disk diffusion assays on Petri dishes
Bharath Srinivasan et al.
European journal of medicinal chemistry, 103, 600-614 (2015-09-29)
Gram-negative bacteria are implicated in the causation of life-threatening hospital-acquired infections. They acquire rapid resistance to multiple drugs and available antibiotics. Hence, there is the need to discover new antibacterial agents with novel scaffolds. For the first time, this study
Marcus S Bray et al.
Proceedings of the National Academy of Sciences of the United States of America, 115(48), 12164-12169 (2018-11-11)
Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life's oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O2, high Fe2+, and high Mn2+) can revert the ribosome to a functional
Wanida Phetsang et al.
ACS infectious diseases, 2(10), 688-701 (2016-10-16)
Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the
Bermans J Iskandar et al.
The Journal of clinical investigation, 120(5), 1603-1616 (2010-04-29)
The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal
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Questions

1–2 of 2 Questions  

  1. 1. what is the origin of this DHFR. Is it from Human, E. coli or any other. 2. I want to relate antibacterial activity of my compound with this enzyme assay kit. Is it obvious.

    1 answer

    1. The Dihydrofolate Reductase that is provided in the kit is product D6566, a recombinant human enzyme that is expressed in E. coli. Please see the link below to review the datasheet for this component:
      https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/213/424/d6566dat.pdf

      One intended use of this kit is for screening DHFR inhibitors, including inhibitors of bacterial DHFR. This kit is expected to be suitable for the measurement of antibacterial activity. This characteristic is expressed as DHFR inhibitory activity of a compound of interest. Please see the link below to review the datasheet for this kit:
      https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/427/408/cs0340bul.pdf

      Helpful?

  2. I want to measure activity in protein variants that have different cellular concentration (protein stability), will this affect my activity?

    1 answer

    1. It is unclear the relation between cellular concentration and protein stability. It seems the intention is to produce dihydrofolate reductase recombinantly and then measure the activity. Protein stability is dependent on several different factors such as pH, temperature, buffering conditions, storage, purification, etc. If a protein is not stable it can certainly affect the activity. Testing would be necessary to determine the overall impact on the activity as you optimize your process.

      To further discuss this application, we kindly ask you to navigate to the link https://www.sigmaaldrich.com/techservice, and click on "Product Technical Inquires" under the Products Section with all the required information so that a member of our team can reach out to you to assist further. Thank you.

      Helpful?

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