B. burgdorferi strains B31A3 and A3HtrAOE were harvested from BSK II medium and washed two times with DPBS. A total of 2 × 107 cells from each were separated by SDS-PAGE and analyzed by western blot using rabbit polyclonal anti-p66, rabbit anti-HtrA and mouse monoclonal anti-FlaB. Secondary antibodies used were goat ant-rabbit IgG IR700 and goat anti-mouse IgG IR800 (Rockland Immunochemicals). Digital images of western blot bands were analyzed using a Bio Rad GD/Chemi Documentation System and Quantity One Quantitation Software (Bio Rad) to determine optical densities. Each band was quantitated three times (left end, middle, right end) and the combined data from two independent experiments was tested for statistical significance by unpaired t test (Graph Pad, La Jolla, CA).
Quantity one quantitation software
Manufactured by Bio-Rad
Sourced in United States
Quantity One Quantitation Software is a laboratory analysis tool developed by Bio-Rad. It provides quantitative analysis of gel-based data, including Western blots and electrophoresis gels.
Automatically generated - may contain errors
Lab products found in correlation
Goat ant rabbit igg ir700, by Rockland Immunochemicals (1 mentions)
Goat anti mouse igg ir800, by Rockland Immunochemicals (1 mentions)
Horseradish peroxidase conjugated secondary antibody against rabbit igg, by Abcam (1 mentions)
Monoclonal anti actin antibody, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Versadoc imaging system, by Bio-Rad (1 mentions)
Gs 710 calibrated imaging densitometer, by Bio-Rad (1 mentions)
Hyperfilm ecl, by Cytiva (1 mentions)
Ecl plus kit, by Cytiva (1 mentions)
Mini protein 3, by Bio-Rad (1 mentions)
Hybond p, by Cytiva (1 mentions)
Sepasol, by Nacalai Tesque (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Redivue α 32p dctp, by Cytiva (1 mentions)
Bacterial genomic dna isolation kit, by Norgen Biotek (1 mentions)
Generuler 100 bp dna ladder, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 system, by Thermo Fisher Scientific (1 mentions)
7 protocols using quantity one quantitation software
1
Western Blot Analysis of B. burgdorferi
2
Western Blot Analysis of ZHX3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This procedure has been described previously.24 ,25 Sixty micrograms of proteins from each sample was subjected to SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After 1-hour incubation in blocking buffer (Tris-buffered saline with 0.1% Tween and 5% nonfat dry milk), the membranes were incubated with a rabbit ZHX3 antibody (Abcam, Cambridge, MA, USA; dilution, 1:500) and a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (Abcam; dilution, 1:1,000). Signals were captured with the enhanced chemiluminescence system following the manufacturer’s instructions (Amersham Pharmacia, Piscataway, NJ, USA). The membranes were reprobed with an anti-actin monoclonal antibody (Abcam; dilution, 1:1,000) to assure the equal loading of each sample. The intensity of ZHX3 was quantified using a Bio-Rad Quantity One quantitation software, with the ratio between the tumor and the paired noncancerous tissues of less than twofolds, suggesting downregulated ZHX3 expression.
3
Lymphocyte Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lymphocytes were isolated from the blood by the method describe previously by Dey et al. [19 (link)]. In brief, after sonication, peripheral blood lymphocytes (PBLs) isolated from case and control were used for immunoblotting. Protein content of the case and control samples was estimated by the method describe by Lowry [20 (link)]. The blood lymphocytes were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 3% stacking gel and 7.5% separating gel) and electroblotted on Immobilon-P membrane (Millipore, USA). The membranes were incubated with the primary antibodies raised against ASPN [21 (link)] and COMP [22 (link)] (1:1000 dilution) in 5 ml of phosphate-buffered saline containing 0.02% Tween-20 and 0.02% sodium azide, PBST for 3 h at room temperature. The membranes carrying lymphocytes were incubated with 1:2000 dilution of rabbit anti-rabbit IgG-horseradish peroxidase (secondary antibody). Following incubation, membranes were washed five times with PBST (5–10 min each) and then processed for detection with chemiluminescence substrate. The membranes were visualized on VERSA DOC Imaging System (Model 1000, Bio-Rad, USA) and quantitative analysis using Quantity One Quantitation software (Bio-Rad). Prior to studying protein expression of these genes, normalization of proteins in the lymphocytes was carried out using beta-actin antibody.
4
Quantification of Hepatic CYP2E1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hepatic CYP2E1 protein expression was determined as described previously [23 (link)]. Briefly, hepatic microsomal proteins (5 μg) were denatured with sample buffer, loaded on a 10 % (w/v) SDS-polyacrylamide gel, and then electrophoresed (Mini-Protein III, Bio-Rad, CA). The gel contents were electrophoretically transferred to a PVDF-membrane (Hybond™-P, Amersham). The membrane was blocked in non-fat skimmed milk and incubated at 4 °C overnight with primary antibody at a 1:20,000 dilution (rabbit anti-rat CYP2E1, Chemicon International, Inc., Temecula, CA). After several washes in 0.1 % Tris Buffered Saline with Tween-20, the membranes were incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Chemicon) at room temperature for 1 h. The ECL Plus kit (Amersham) was used for the chemiluminescence reaction and exposure to Hyperfilm ECL™ (Amersham). GS-710 Calibrated-Imaging densitometer and Quantity One Quantitation software (Bio-Rad, Hercules, CA) were used for CYP2E1 protein analysis.
5
Gene Expression Analysis by RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using Sepasol (Nacalai Tesque) with manufactural instructions. 2 µg of RNA were used for cDNA synthesis with ReverTra Ace (Toyobo, Tokyo, Japan). The sequences of primers (Invitrogen, Carlsbad, CA, USA) for analyzed genes were; 5'-ACCACAGTCCATGCCATCAC-3' and 5'-CACCACCCTGTTGCTGTAGCC-3' for GAPDH, 5'-GAGCCCCGGGGTGGAACAAGAT-3' and 5'-AAAAGGTGGTGGGCAGGAGTAA-3' for ATGL, 5'-CCTACTGCTGGGCTGTCAA-3' and 5'-CCATCTCGCACCCTCACT-3' for HSL. The PCR program was 30 cycles of denaturation at 98℃ for 10 seconds, annealing at 58-60℃ for 30 seconds and extension at 68℃ for 30 seconds by iCycler (Bio-Rad, Munich, Germany). The products were visualized by ethidium bromide agarose gels and the images were detected with UV irradiation. The densitometry result for target samples was divided by the densitometry result of GAPDH, a constitutive gene control, normalizing the level considered for analysis. Densitometry was carried out using a Bio-Rad ChemiDoc image acquisition system and Quantity One quantitation software (Bio-Rad, Hercules, CA, USA).
6
Quantitative RT-PCR Protocol for Plant Samples
7
RAPD Genotyping of Pseudomonas aeruginosa
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
P. aeruginosa isolates were grown in 5 ml Pseudomonas Selective Broth (Biolife, Milan, Italy) with incubation at 35°C overnight. DNA was then extracted from bacterial pellets using the Bacterial Genomic DNA Isolation Kit (Norgen Biotek, Thorold, Canada) and its concentration estimated through the NanoDrop ND-1000 System (NanoDrop Technologies, Wilmington, DE). RAPD (Random Amplification of Polymorphic DNA) typing was carried out according to Lanotte et al. [26 (link)] with 272 primer (5’-AGCGGGCCAA-3’) and 40 ng DNA of each strain. PCR products (12 μl) were run on 1.5% agarose gel with GeneRuler 100 bp DNA Ladder (Thermo Scientific) and analyzed on a Gel Doc 2000 apparatus using Quantity One Quantitation Software (Bio-Rad, Hercules, CA, USA). The molecular size (bp) of each potential band position was determined across all RAPD-PCR profiles. At each band position, two possible alleles were considered either present (a score of 1) or absent (a score of 0). Different RAPD profiles were designated by different scores and classified as different genotypes.
A dendrogram was constructed through the MVSP software (ver. 3.22) (available athttp://mvsp.software.informer.com/3.2/ ). After construction of a similarity matrix, data were cluster analyzed using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method with similarity of distance according Jaccard’s coefficient.
A dendrogram was constructed through the MVSP software (ver. 3.22) (available at
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!