Recombinant Rat POLA1 Protein

• Cat.No.: POLA1-4560R
• Product Overview: Recombinant Rat POLA1 full length or partial length protein was expressed.

Specification

• Species: Rat
• Source: Mammalian Cells
• Tag: His
• Form: Liquid or lyophilized powder
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method.
• Purity: >80%
• Notes: This item requires custom production and lead time is between 5-9 weeks. We can custom produce according to your specifications.
• Storage: Store it at +4 ºC for short term. For long term storage, store it at -20 ºC~-80 ºC.
• Storage Buffer: PBS buffer

Gene Information

• Gene Name: Pola1 polymerase (DNA directed), alpha 1, catalytic subunit [ Rattus norvegicus ]
• Official Symbol: POLA1
• Gene ID: 85241
• mRNA Refseq: NM_053479.1
• Protein Refseq: NP_445931.1
• UniProt ID: O89042

Citation

Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

Journal: American Journal of Cancer Research    PubMed ID: 29416919    Data: 2018/1/1

Authors: Rana Abdel-Samad, Patrick Aouad, Nadine Darwiche

Article Snippet:The final concentrations used were 0, 0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM.The final concentrations used were 0, 0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM.. One microliter of diluted compounds was added to 1 unit of the full length POLA1 recombinant protein (Creative BioMart, Shirley, NY), 2 μl of 10 mg/ml BSA, and 0.4 μl of 2.5 mM dNTP and pre-incubated at 25°C for 15 minutes.. Afterwards, 3 μl of the primer extension substrate was added to the mixture.Afterwards, 3 μl of the primer extension substrate was added to the mixture.

Forward and reverse primer sequences

ST1926-resistant cells are cross-resistant to CD437 and both compounds inhibit POLA1 activity. A. Effect of ST1926 treatment on the growth of human HCT116-STR. ST1926-resistant cells (HCT116-STR) were generated by treating the parental cell line with increasing concentrations of ST1926 over a period of eight months. HCT116-STR cells were seeded in 96-well plates at a density of 5,000 cells/well, and treated with the indicated concentrations of ST1926 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of three independent experiments ± SEM. B. HCT116-STR DNA damage response to ST1926 treatment. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to 24 hours. Total SDS protein lysates (50 μg/lane) were immunoblotted against γ-H2AX antibody. Similar trends in protein levels were observed in three independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. TUNEL analysis of HCT116-STR cells treated with 0.1% DMSO or 1 μM ST1926 up to 48 hours. Results are representative of two independent experiments. D. Effect of CD437 treatment on HCT116 and HCT116-STR cell growth. Cells were treated with the indicated concentrations of CD437 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of at least three independent experiments ± SEM. E. Primer extension assay was evaluated in the presence of increasing concentrations of ST1926 and CD437 (0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM). “-” represents the control (DMSO). The activity of POLA1 was determined by the detection of primer of a 25-nucleotide product, and a gel representative of two independent experiments is shown.

HCT116-resistant ST1926 cells exclusively harbor two missense mutations in POLA1. A. Schematic representation of POLA1 mutations identified in ST1926-resistant HCT116 cells (HCT116-STR). RNA was extracted from HCT116-parental and HCT116-STR cells and reverse transcribed, POLA1 cDNA was sequenced by the Sanger method. The panels of DNA sequencing were compared and relevant regions in exon 21 of POLA1 are shown. B. Effect of 5-fluorouracil (5-FU) treatment on the growth of HCT116 and 5-FU resistant (HCT116-FUR) cells. Cells were seeded in 96-well plates at a density of 5,000 cells/well, and treated with the indicated concentrations of 5-FU for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of at least three independent experiments ± SEM. C. DNA sequences were translated using the Multiple alignment server (MUSCLE) and protein sequences were compared in HCT116, HCT116-STR, and HCT116-FUR cells. Only relevant regions of the sequences are shown.