RPA1 ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Replication Protein A1 (RPA1) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Replication Protein A1 (RPA1) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

HSSB; MST075; MSTP075; REPA1; replication factor A protein 1; replication protein A 70 kDa DNA-binding subunit; replication protein A1, 70kDa; RF-A; RF-A protein 1; RP-A; RP-A p70; RPA70; single-stranded DNA-binding protein.

As part of the heterotrimeric replication protein A complex (RPA/RP-A), binds and stabilizes single-stranded DNA intermediates, that form during DNA replication or upon DNA stress. It prevents their reannealing and in parallel, recruits and activates different proteins and complexes involved in DNA metabolism (PubMed:27723720, PubMed:27723717) . Thereby, it plays an essential role both in DNA replication and the cellular response to DNA damage (PubMed:9430682) . In the cellular response to DNA damage, the RPA complex controls DNA repair and DNA damage checkpoint activation. Through recruitment of ATRIP activates the ATR kinase a master regulator of the DNA damage response (PubMed:24332808) . It is required for the recruitment of the DNA double-strand break repair factors RAD51 and RAD52 to chromatin in response to DNA damage (PubMed:17765923) . Also recruits to sites of DNA damage proteins like XPA and XPG that are involved in nucleotide excision repair and is required for this mechanism of DNA repair (PubMed:7697716) . Plays also a role in base excision repair (BER) probably through interaction with UNG (PubMed:9765279) . Also recruits SMARCAL1/HARP, which is involved in replication fork restart, to sites of DNA damage. May also play a role in telomere maintenance (PubMed:17959650) . As part of the alternative replication protein A complex, aRPA, binds single-stranded DNA and probably plays a role in DNA repair. Compared to the RPA2-containing, canonical RPA complex, may not support chromosomal DNA replication and cell cycle progression through S-phase. The aRPA may not promote efficient priming by DNA polymerase alpha but could support DNA synthesis by polymerase delta in presence of PCNA and replication factor C (RFC), the dual incision/excision reaction of nucleotide excision repair and RAD51-dependent strand exchange (PubMed:19996105) .

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Replication Protein A1 (RPA1) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Replication Protein A1 (RPA1) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Replication Protein A1 (RPA1), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of Replication Protein A1 (RPA1) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Component Amount Anti-RPA1 Microplate 96 Wells (12 x 8 Well Strips) RPA1 Lyophilized Standard 2 x 20 ng 100X Biotinylated RPA1 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-RPA1 Microplate 96 Wells (12 x 8 Well Strips) Anti-RPA1 Microplate 96 Wells (12 x 8 Well Strips) RPA1 Lyophilized Standard 2 x 20 ng RPA1 Lyophilized Standard 2 x 20 ng 100X Biotinylated RPA1 Detector Antibody 120 uL 100X Biotinylated RPA1 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Replication Protein A1 (RPA1) . No significant cross-reactivity or interference between Replication Protein A1 (RPA1) and analogues was observed.