Journal: BMC Biotechnology

Article Title: A simple, accurate and universal method for quantification of PCR

doi: 10.1186/s12896-016-0256-y

Figure Lengend Snippet: Quantification of PCR amplified nucleic acid using AccuCal calibrator. a The workflow associated with using AccuCal to quantify input nucleic acid amount in each PCR, b AccuCal calibrator was diluted so that 0, 40, 60, 80, 120, 140 and 200 ng in Sso Fast EvaGreen Supermix were added to respective wells of a PCR plate and subjected to 40 amplification cycles. c The fluorescence intensity of each AccuCal calibrator (after subtraction of mean 0 ng AccuCal fluorescence) was plotted against the amount (pmols) and a linear regression line fitted to generate the calibration curve. d Ten-fold dilutions of lambda DNA, ranging from 4.5 × 10 6 – 4.5 × 10 1 copies/PCR, were amplified in quadruplicate in Sso Fast EvaGreen Supermix on the same plate as the AccuCal calibrators. e The calibration curve was used, alongside the calculated efficiency of each amplification reaction and the cycle numbers between the take-off point (Cq) and second derivative maxima for each amplification reaction, to quantify the mean starting amount of DNA in each PCR. The standard error of the mean is also presented. f The theoretical and determined number of copies/PCR, plus SEM, were plotted against each other and a regression line drawn to demonstrate the agreement between the two values

Article Snippet: RNA was reverse transcribed using qScript cDNA supermix (Quanta Biosciences) according to the manufacturer’s instructions.

Techniques: Amplification, Fluorescence, Lambda DNA Preparation

Journal: The Journal of investigative dermatology

Article Title: Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.

doi: 10.1046/j.1523-1747.1998.00413.x

Figure Lengend Snippet: Figure 1. Characterization of the Hom s 1 cDNA. (a) cDNA and deduced amino acid sequence of Hom s 1. Clone a (start and end indicated by arrows) was isolated by IgE immunoscreening of a A431 cDNA expression library. cDNA clones b and c (start and end indicated by solid and open arrowheads) were obtained by oligonucleotide screening of a cDNA library from human testis. A Sac I site (GAGCTC) in the Hom s 1 cDNA is underlined and printed in italics. The putative start signal (ATG) is printed in bold letters and the polyadenylation signal (aataaa) is underlined and in italics. The deduced amino acid sequence of Hom s 1 contains: (i) a leucine zipper motif (LEEIRAKLRLQAQSLSTVGP) (underlined, broken line); (ii) two amidation sites (italics, underlined, solid line); (iii) two tyrosine kinase interaction sites (underlined, solid line); (iv) two N-glycosylation positions (bold letters, underlined, solid line); and (v) a potential glycosaminoglycan-binding domain (underlined, dotted line). The cDNA and deduced amino acid sequences of Hom s 1 have been deposited in the EMBL library under the accession number Y14314. (b) Southern blot hybridization of A431 genomic DNA with the Hom s 1 cDNA. Twenty microgram aliquots of human genomic DNA were digested with either Sac I (lane S), Hind III (lane H), or Bam H I (lane B), separated by 1% agarose gel electrophoresis, and blotted onto nitrocellulose. For control purposes, 5 µg of Pst I-digested lambda DNA was used (lane M). The positions of the molecular weight markers are shown on the left. The blot was hybridized with the 32P-dCTP-labeled cDNA of clone a. Hybridization of the Sac I- digested genomic DNA resulted in two bands (asterisks) because of one Sac I site (a, italics, underlined) in the Hom s 1 gene. (c) Northern blot. Twenty micrograms of nitrocellulose-blotted HMC-1 RNA were hybridized with the 32P-dCTP-labeled cDNA of clone a. The positions of the 28S and 18S ribosomal RNA are indicated on the left side of the blot.

Article Snippet: Cell lines, tissue specimens, anti-sera The human epithelial cell line A431, established from an epidermoid mammary carcinoma, was obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Sequencing, Isolation, Expressing, Clone Assay, cDNA Library Assay, Glycoproteomics, Binding Assay, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Control, Lambda DNA Preparation, Molecular Weight, Labeling, Northern Blot

Journal: The Journal of investigative dermatology

Article Title: Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.

doi: 10.1046/j.1523-1747.1998.00413.x

Figure Lengend Snippet: Figure 2. IgE binding capacity of recombinant Hom s 1. (a) Serum IgE from AD patients recognizes recombinant Hom s 1. Recombinant Hom s 1 was expressed as a β-gal fusion protein in E. coli Y1089. Approximately 5 µg per cm gel of the purified recombinant proteins were separated by 12.5% SDS- PAGE and blotted onto nitrocellulose. Lane C shows sections of Coomassie- Blue-stained gels containing the purified proteins. Nitrocellulose-blotted Hom s 1-β-gal fusion protein or β-gal alone was incubated with sera from AD patients (lanes 1–15), serum from a nonallergic individual (lane N), buffer without serum (lane B), or a supernatant from the human IgE-myeloma cell line U266 (Nilsson et al, 1970) (lane M). Bound IgE was detected with 125I- labeled anti-IgE antibodies. The total IgE levels (kU per liter) (CAP, Pharmacia) of patients’ sera and of the U266 supernatant were as follows: lane 1, 2050; 2, 1965; 3, 15,350; 4, 12,560; 5, 12,980; 6, 1416; 7, 14; 8, 35; 9, 6180; 10, 133; 11, 19; 12, 12,500; 13, 1756; 14, 1466; 15, 2100; N, 7; M, 1300. (b) Recombinant Hom s 1 specifically inhibits the binding of Hom s 1-specific serum IgE to an A431-derived 55 kDa protein. A 1:10 diluted serum was preabsorbed with 5 µg purified Hom s 1-β-gal(a) or β-gal(β) per ml prior to exposure to nitrocellulose-blotted A431 extracts, and bound IgE was detected.

Article Snippet: Cell lines, tissue specimens, anti-sera The human epithelial cell line A431, established from an epidermoid mammary carcinoma, was obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Binding Assay, Recombinant, SDS Page, Staining, Incubation, Labeling, Derivative Assay