Impact of HIV subtype on performance of the limiting antigen-avidity enzyme immunoassay, the bio-rad avidity assay, and the BED capture immunoassay in Rakai, Uganda.

Impact of HIV subtype on performance of the limiting antigen-avidity enzyme immunoassay, the bio-rad avidity assay, and the BED capture immunoassay in Rakai, Uganda.

Previous research demonstrated that people with subtype D HIV an infection who had been contaminated for two or extra years have been incessantly misclassified as assay optimistic utilizing cross-sectional incidence assays.

Samples from 510 topics (212 subtype A, 298 subtype D) who have been contaminated for two.2 to 14.5 years (median 5.Four years) and weren’t virally suppressed have been examined utilizing an LAg-Avidity enzyme immunoassay (LAg-Avidity EIA), Bio-Rad Avidity assay, and BED capture enzyme immunoassay (BED-CEIA).

The performance of these three assays was evaluated utilizing numerous assay cutoff values [LAg-Avidity EIA: <1.0 OD-n and <2.0 OD-n; Bio-Rad Avidity assay: <40% avidity index (AI) and <80% AI; BED-CEIA: <0.8 OD-n]. The imply LAg-Avidity EIA consequence was increased for subtype A than D (4.54±0.95 vs. 3.86±1.26, p<0.001); the imply Bio-Rad Avidity assay consequence was increased for subtype A than D (88.9%±12.5% vs. 75.1±30.5, p<0.001); and the imply BED-CEIA consequence was related for the two subtypes (2.2±1.2 OD-n for subtype A, 2.2±1.Three OD-n for subtype D, p<0.9).

The frequency of misclassification was increased for people with subtype D an infection in comparison with these with subtype A an infection, utilizing both the LAg-Avidity EIA with a cutoff of <2.Zero OD-n or the Bio-Rad Avidity assay with cutoffs of <40% or <80% AI.

No subtype-specific variations in assay performance have been noticed utilizing the BED-CEIA. Sex and age weren’t considerably related to misclassification by any assay. The LAg-Avidity EIA with a cutoff <1.Zero OD-n had the lowest frequency of misclassification in this Ugandan inhabitants.

Impact of HIV subtype on performance of the limiting antigen-avidity enzyme immunoassay, the bio-rad avidity assay, and the BED capture immunoassay in Rakai, Uganda.
Impact of HIV subtype on performance of the limiting antigen-avidity enzyme immunoassay, the bio-rad avidity assay, and the BED capture immunoassay in Rakai, Uganda.

A bio-bar-code assay primarily based upon dithiothreitol-induced oligonucleotide launch.

The just lately developed bio-bar-code assay for the PCR-less detection of protein and nucleic acid targets has been proven to be terribly delicate, exhibiting low attomolar sensitivity for protein targets and excessive zeptomolar sensitivity for nucleic acid targets. In the case of DNA detection, the unique assay depends on three distinct oligonucleotide strands on a single nanoparticle for goal identification and sign amplification.

Herein, we report the growth of a brand new nanoparticle probe that can be utilized in the bio-bar-code assay, which requires just one thiolated oligonucleotide strand. This new assay depends on the capacity to liberate the adsorbed thiolated oligonucleotides from the gold nanoparticle floor with dithiothreitol (DTT), which simplifies the assay and will increase its quantitative capabilities.

The utility of this new DTT-based system is demonstrated by detecting a mock mRNA goal utilizing each fluorescent and scanometric assay readouts. When the scanometric readout is used, the sensitivity of the assay is 7 aM and quantification will be achieved over the low-attomolar to the mid-femtomolar focus vary.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

Advances within the growth of extremely multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are mentioned. Use of novel reagents particularly designed for immunological methods using elemental evaluation is introduced.

The main steps of methodology growth, together with choice of components for tags, validation of tagged reagents, and examples of multiplexed assays, are thought of intimately.

The paper additional describes experimental protocols for elemental tagging of antibodies, immunostaining of reside and stuck human leukemia cells, and preparation of samples for ICP-MS evaluation.

Quantitative evaluation of floor antigens on mannequin cell traces utilizing a cocktail of seven lanthanide labeled antibodies demonstrated excessive specificity and concordance with standard immunophenotyping.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.
Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery.

Bioluminescence-based assays to point antimicrobial susceptibility have been developed and validated for recombinant strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, and Mycobacterium intracellulare expressing an built-in eukaryotic luciferase gene.

MICs decided with these bioluminescence assays for a number of antimycobacterial brokers, together with isoniazid, ethambutol, rifampin, amikacin, streptomycin, ciprofloxacin, and clarithromycin, in contrast favorably with conventional BACTEC methods and visible estimations of the inhibitory finish level. Assay methodology has been optimized for the evaluation of giant numbers of novel compounds and is easy, cheap, and labor environment friendly.

The availability of these 4 recombinant mycobacteria has permitted a technique for drug discovery using the nonpathogenic BCG pressure for mass screening functions with subsequent affirmation of exercise towards the pathogenic mycobacteria. Furthermore, proof means that the BCG-based display could permit the direct identification of bactericidal brokers.

Comparison of serum folate species analyzed by LC-MS/MS with complete folate measured by microbiologic assay and Bio-Rad radioassay.

BACKGROUNDThe Bio-Rad QuantaPhase II radioassay (BR), used for 25 years to measure complete folate (TFOL) concentrations for the National Health and Nutrition Examination Survey (NHANES), will likely be discontinued in 2007. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) or a microbiologic assay (MA) will likely be used sooner or later.

Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy.

Nanoparticle-based bio-barcode assay redefines "undetectable" PSA and biochemical recurrence after radical prostatectomy.

We report the event of a beforehand undescribed gold nanoparticle bio-barcode assay probe for the detection of prostate particular antigen (PSA) at 330 fg/mL, automation of the assay, and the outcomes of a scientific pilot examine designed to evaluate the flexibility of the assay to detect PSA within the serum of 18 males who’ve undergone radical prostatectomy for prostate most cancers.

Due to an absence of sensitivity, accessible PSA immunoassays are sometimes not able to detecting PSA within the serum of males after radical prostatectomy. This new bio-barcode PSA assay is roughly 300 instances extra delicate than business immunoassays.

Significantly, with the barcode assay, each affected person on this cohort had a measurable serum PSA degree after radical prostatectomy. Patients had been separated into classes primarily based on PSA ranges as a operate of time.

One group of sufferers confirmed low ranges of PSA with no important enhance with time and didn’t recur. Others confirmed, sooner or later postprostatectomy, rising PSA ranges. The majority recurred.

Therefore, this new ultrasensitive assay factors to important potential outcomes: (i) The capacity to inform sufferers, who’ve undetectable PSA ranges with typical assays, however detectable and nonrising ranges with the barcode assay, that their most cancers won’t recur. (ii) The capacity to assign recurrence earlier due to the flexibility to measure growing ranges of PSA earlier than typical instruments could make such assignments. (iii) The capacity to make use of PSA ranges that aren’t detectable with typical assays to comply with the response of sufferers to adjuvant or salvage therapies.

Nanoparticle-based bio-barcode assay redefines "undetectable" PSA and biochemical recurrence after radical prostatectomy.
Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy.

Neutralization assays for differential henipavirus serology utilizing Bio-Plex protein array methods.

Hendra virus (HeV) and Nipah virus (NiV) are associated rising paramyxoviruses categorised within the genus Henipavirus. Both trigger deadly illness in animals and people and are categorised as biosafety degree four pathogens.

Here we element two new multiplexed microsphere assays, one for antibody detection and differentiation and one other designed as a surrogate for virus neutralization. Both assays make the most of recombinant soluble attachment glycoproteins (sG) whereas the latter incorporates the mobile receptor, recombinant ephrin-B2.

Spectrally distinct sG(HeV)- and sG(NiV)-coupled microspheres preferentially sure antibodies from HeV- and NiV-seropositive animals, demonstrating a easy process to distinguish antibodies to those carefully associated viruses.

Soluble ephrin-B2 sure sG-coupled microspheres in a dose-dependent vogue. Specificity of binding was additional evaluated with henipavirus G-specific sera and MAbs. Sera from henipavirus-seropositive animals differentially blocked ephrin-B2 binding, suggesting that detection and differentiation of HeV and NiV neutralizing antibodies will be achieved concurrently within the absence of reside virus.

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3MM Whatman paper
Whatman, Dassel, Germany
Autoclave
Integra Biosciences, Baar, Switzland
Benchtop gyroory incubator
New Brunswick Scientific, Edison
shaker for bacterium
N.J. USA
Bacteria incubator
Heraeus, Hanau, Germany
Bandelin Sonopuls HD 2070
South Yorkshire, England
Cell culture flasks/dishes
Greiner, Heidelberg, Germany
Cell culture incubator
Heraeus, Hanau, Germany
CELL locate glass cover slips with grids,
Eppendorf, Hamburg, Germany
– Grid size 55 μm
Cell scraper
Corning, Wiesbaden, Germany
Cell strainer (40μm)
Becton Dickinson, Heidelberg, Germany
FACS (Fluorescence-activated cell sorting)
FACScan, Beckton Dickinson, Heidelberg
Centrifuge
Cytocentrifuge
Thermo Shandon, Pittsburgh, USA
Rotina 48 RS Table top centrifuge
Hettich, Tuttlingen, Germany
-Sorvall RC- 5B refrigerated superspeed
centifuge and Biofuge 13R
Heraeus, Hanau, Germany
Table top centrifuge 5415C
Eppendorf, Hamburg, Germany
Megafuge 1.0 with Rotor BS4402/A
Heraeus, Hanau, Germany
Chemiluminescence detection
-Film BioMax
Eastman-Kodak, Rochester, USA
Corning 125 pH meter
Fisher Scientific, Shwerte, Germany
Cryotubes
Nalgene, Rochester, NY , USA
Cytofunnel
Shandon, Pittsburgh, USA
Electroporation:
Gene Pulser System with electroporation
Bio-Rad Laboratories, Munich, Germany
Cuvette 0.4 cm
Flow Chamber Kit
-Circular parallel plate
GlycoTech, Maryland, U.S.A
Gel electrophoresis system
-DNA-sub cell and Mini-Sub cell System
Gibco, Betheseida, USA
Power PAC 2000
BioRad, Munich, Germany
Irradiation of mice
-Betatron 500A
Siemens, Munich, Germany
Lumat LB 9507
Berthold, Bad Wildbad, Germany
Magnetic cell separation
-MiniMACS separator
Miltenyi, Bergisch Gladbach, Germany
-MidiMACS separator
Miltenyi, Bergisch Gladbach, Germany
-MACS separation columns: LD and LS
Miltenyi, Bergisch Gladbach, Germany
Microscope
-SZ40 Zoom Stereo Microscope
Olympus, Munich, Germany
Axioplan II-imaging Fluorescence microscope
Zeiss, Goettingen, Germany
Leitz Labovert inverted Microscope
Leitz, Wetzlar, Germany
Zeiss ID03 inverted microscope
Zeiss, Goettingen, Germany
Nitrocellulose membrane (0.45 μm)
Bio-Rad Laboratories, Munich, Germany
PCR
-Techne TC-312
Kisker , Steinfurt, Germany
Protein transfer
-Trans-Blot SD Semi-dry Transfer cell
BioRad, Munich, Germany
Spectrohotometer
-Gene Quant II
Pharmacia Biotech, Freiburg,
-Ultraspec® 2000
Germany
Steril bank
Heraeus, Hanau, Germany
Steril filter
-0.22μM
Millipore, Eschborn, Germany
-0.45μM
Millipore, Eschborn, Germany
Vortex
Eppendorf, Hamburg, Germany
Water bath
Julabo Labortechnik, Seelbach, Germany
Acetic acid glacia
Sigma, Steinheim, Germany
Acetone
Roth, Karlsruhe, Germany
Ammonium persulfate
Fluky, Deisenhofen, Germany
Ampicillin
Sigma, Steinheim, Germany
Anti-Sca-1 Microbeads
Miltenyi, Bergisch Gladbach, Germany
Biorad protein dye
Biorad, Munich, Germany
Borax
Sigma, Steinheim, Germany
BrdU
Sigma, Steinheim, Germany
β-Mercaptoethanol
Fluka , Deisenhofen, Germany
Bromophenol blue
Sigma, Steinheim, Germany
Calcium chloride
Sigma, Steinheim, Germany
Chlorofor
Fluka , Deisenhofen, Germany
Cumaric acid
Sigma, Steinheim, Germany
ddH2O
Sigma, Steinheim, Germany
DEPC
Sigma, Steinheim, Germany
DMSO
Sigma, Steinheim, Germany
EDTA, disodium, dihydrate
Sigma, Steinheim, Germany
Ethanol
Merck, Darmstadt, Germany
Ethidiumbromid
Sigma, Steinheim, Germany
Formaldehyd
Merck, Darmstadt, Germany
Forene (Isofluran)
Abbott GmbH, Wiesbaden, Germany
Glucose
Sigma, Steinheim, Germany
Glutathione sepharose
TM
Amersham/Pharmacia Biotech, Freiburg,
Germany
Glycerol
Sigma, Steinheim, Germany
Glycin
Merck, Darmstadt, Germany
Isopropylthio-β-D-Galactoside (IPTG)
Sigma, Steinheim, Germany
HCL
Merck, Darmstadt, Germany
Isobutanol
Sigma, Steinheim, Germany
Isopropanol
Fluka , Deisenhofen, Germany
Kanamycin
Sigma, Steinheim, Germany
Lineage cell depletion kit
Miltenyi, Bergisch Gladbach, Germany
Luminol
Fluka , Deisenhofen, Germany
Sigma, Steinheim, Germany
MgSO4
Fluka , Deisenhofen, Germany
NaN3
Sigma, Steinheim, Germany
NaCl
Merck, Darmstadt, Germany
NaH2PO4
Sigma, Steinheim, Germany
NaOH
Roth GmbH, Karlsruhe, Germany
Natriumacetat
Fluka , Deisenhofen, Germany
Paraformaldehyd
Merck, Darmstadt, Germany
p-Coumaric acid
Sigma, Steinheim, Germany
PD-10 columns
Amersham/Pharmacia Biotec, Freiburg,
Germany
Phenol
Fluka , Deisenhofen, Germany
PKH26 Red fluorescence kit
Sigma, Steinheim, Germany
Polybrene
Sigma, Steinheim, Germany
Ponceau S
Sigma, Steinheim, Germany
Propedium iodide
Sigma, Steinheim, Germany
Protease-inhibitors
Roche Basel, Switzerland
Pre-stained SDS-PAGE standards
-Broad range
Bio-Rad Laboratories, Munich, Germany
Retronectin
®
Takara Bio Inc., Otsu, Japan
Sca-1 Isolation Kit
Miltenyi, Bergisch Gladbach, Germany
SDS
Sigma, Steinheim, Germany
STREPtactin sepharose
TM
IBA, Goettingen, Germany
Protein elution buffer
IBA, Goettingen, Germany
TEMED
Sigma, Steinheim, Germany
Tritriplex III, 1,1%
University teaching hospital Frankfurt
Trizma HCl
Sigma, Steinheim, Germany
Triton X-100
Sigma, Steinheim, Germany
Tween20
Sigma, Steinheim, Germany
Vivapore (For concentrating protein)
VivaScience, Stonehouse, UK
Xylencyanol
Sigma, Steinheim, Germany
7-aminoactinomycin D (7-AAD)
Sigma, Steinheim, Germany
BSA, V solution
Sigma, Steinheim, Germany
Chloroquine
Sigma, Steinheim, Germany
DMEM, -High glucose (4.5g/l)
Invitrogen, Karlsruhe, Germany
DMSO
Sigma, Steinheim, Germany
FBS
Gibco BRL, Paisley, Schottland
Ficoll separating solution
Biochrom (# L-6115), Berlin, Germany
Gelatin
Sigma, Steinheim, Germany
GNF-2
Sigma, Steinheim, Germany
HBSS
Gibco, Karlsruhe, Germany
HEPES solution
Gibco, Karlsruhe, Germany
Horse serum
Gibco, Karlsruhe, Germany
Imatinib
Novartis, Basel Switzerland
IMD
Gibco, Karlsruhe, Germany
L-Glutamine
Gibco, Karlsruhe, Germany
Methoculttm GF M3434
StemCell Technologies, Vancouver,
Canada
Mycosa
Gibco, Karlsruhe, Germany
PBS
Gibco, Karlsruhe, Germany
Penicillin-Streptomycin solution
Gibco, Karlsruhe, Germany
Retinoic acid
Sigma, Steinheim, Germany
Retronectin
Takara, Shiga, Japan
RPMI 1640
Gibco, Karlsruhe, Germany
Trypan blue stain (0.4%)
Gibco, Karlsruhe, Germany
Trypsin EDTA, 0.25% solution
Gibco, Karlsruhe, Germany
rmG-CSF
Peprotech, Offenbach, Germany
rmSCF
Peprotech, Offenbach, Germany
rmIL-6
Peprotech, Offenbach, Germany
rmIL-3
Peprotech, Offenbach, Germany
rmGM-CSF
Peprotech, Offenbach, Germany
Calf intestinal phosphatase (CIP)
NEB, Frankfurt, Germany
Gateway LR clonase enzyme mix
Invitrogen, Karslruhe, Germany
Klenow-Fragment DNA-polymerase I
NEB, Frankfurt, Germany
Restriction endonucleases
NEB, Frankfurt, Germany
RNAse
Sigma, Steinheim, Germany
Superscript II
Invitrogen, Karlsruhe, Germany
T4 DNA-ligase
NEB, Frankfurt, Germany
Taq-DNA-polymerase
Biosystems, Weiterstadt, Germany
Proteinase K
Stratagene, La Jolla, USA
Taq-DNA-polymerase
Biosystems, Weiterstadt, Germany
DNTPs
Fermentas, St. Leon-Rot, Germany
PCR Buffer
Fermentas, St. Leon-Rot, Germany
MgCl2
Fermentas, St. Leon-Rot, Germany
PfU turbo polymerase
Stratagene, La Jolla, USA
Primers
Sigma, Steinheim, Germany
Mouse anti-β-tubulin (Ab-4)
Neo Markers, Asbach, Germany
Mouse lineage panel
Pharmingen, San Diego, CA, USA
Rabbit anti-c-ABL (11/24)
Santa Cruz, Heidelberg, Germany
Rabbit anti-p-ABL-Tyr-245
Upstate-Biotechnology, Lake Placid, NY,
USA
Rabbit anti-p-ABL-Tyr-412
Cell Signalling, Boston, USA
Rabbit anti-p-BCR-Tyr-177
Cell Signalling, Boston, USA
Rabbit anti-BCR(C-20)
Santa Cruz, Heidelberg, Germany
Mouse anti- GFP
Upstate-Biotechnology, Lake Placid, NY,
USA
Mouse anti-phospho-STAT5
Cell Signalling, Boston, USA
Rabbit anti -STAT5
Cell Signalling, Boston, USA
Rabbit anti-phospho-CrkL (Tyr207)
Cell Signalling, Boston, USA
Mouse anti-CrkL
Cell Signalling, Boston, USA
Mouse anti-phosphotyrosine (clone 4G10)
Upstate-Biotechnology, Lake Placid, NY,
USA
Anti-mouse IgG-HRP
Santa Cruz, Heidelberg, Germany
Anti-rabbit IgG-HRP
Santa Cruz, Heidelberg, Germany
Mouse-anti-human-PE-LNGFR
BD, San Jose, CA, USA
Rat-anti- mouse-PE-Gr-1
BD, San Jose, CA, USA
Rat-anti- mouse-PE-Mac-1
BD, San Jose, CA, USA
Rat-anti- mouse-PE-B220
BD, San Jose, CA, USA
Mouse-anti-PE-IgG 2a, K
BD, San Jose, CA, USA
Mouse-anti-PE-IgG 2b, K
BD, San Jose, CA, USA
Rat –anti-Mouse FITC-Sca-1
BD, San Jose, CA, USA
Rat-anti- mouse-PE-Sca-1
BD, San Jose, CA, USA
MACS buffer
20% (w/v) BSA
12.5 ml
EDTA
1mmol
Penicillin/Streptomycin
5ml
Add PBS up to
500ml
2M CaCl2 solution
The component was dissolved in ddH2O and sterile filtered with 0.22 μm filter and stored at –
20°C in aliquots until use.
2X HBS solution
Na2HPO4
0.315g (1.5 mM final)
HEPES
19.5g (0.05M final)
NaC
24g (0.28M final)
Water added up to 1200ml, three clean bottles were filled with exactly 400 ml of the solution,
the pH to 6,95/ 7,0 / 7,05 was adjusted respectively, each bottle was filled to 500 ml, pH was
controlled again. The buffer was filter-sterilized and stored at -20°C in aliquots.
1x TAE Electrophoresis buffer
Tris-Acetate
0,04M
EDTA
1 mM
Buffer W for lysis of bacteria
Tris-HCl pH 8.0
100 mM
NaCl
150 mM
EDTA
1 mM
Tween
0.1%
Protease inhibitor
1x
1X SDS gel-loading buffer
Tris-HCl, pH 6.8
50mM
SDS
2 % (w/v)
Glycerol
10% (v/v)
β-mercaptoethanol
100mM
Bromophenol blue
0.1 %( v/v)
1X SDS gel-loading buffer lacking dithiothretitol was stored at room temperature. β-
mercaptoethanol from a 14 M stock was added just before the buffer is used.
For protein electrophoresis
(Invitrogen life technologies, Germany)
NuPAGE Novex Bis-Tris
Tris-Acetate Pre-Cast Gels
NuPAGE Novex MES and Tris-Acetate SDS Running Buffers
NuPAGE Transfer Buffer
Precision plus protein (PPP)
NuPAGE LSD Sample Buffer
NuPAGE Reducing Agent
NuPAGE Antioxidant
10X TBS
Trizma HCl, pH 7.5
volume was adjusted to 1 L with wate.
TBST
1X TBS+ 0.05% Tween 20
Blocking Buffer for immunoblot
5% Carnation non-fat dry milk in TBST
Protein transfer buffer
TrisHCl
48 mM
Glycine
Coomassie staining solution
Coomassie brilliant blue R250
0.1 % (w/v)
Methanol
50 % (v/v)
Glacial acetic acid
10 % (v/v)
Coomassie destaining Solution
Methanol
50 % (v/v)
Glacial acetic acid
10 % (v/v)
Ponceau S staining solution
Ponceau S
2 % (w/v)
Trichloroacetic acid
30 % (v/v)
Sulfosalicylic acid.
30 % (v/v)
ECLsolution I:
1 M TrisHCl, pH 8,5
10ml
90mM p-Coumaric acid (in DMSO) 0.44ml
250mM Luminol (in DMSO) 1ml
Water was dded up to 100ml; aliquots were made and stored in the dark at -20°C.
ECLsolution II:
30 % H2O2
64μl
1 M TrisHCl, pH 8,5
10 ml
Water was added up to 100ml, aliquat solution and store in the dark at -20°C.
Stripping buffer
Tris-HCl, pH8.0
62.5 mM
2-mercaproethanol
100 mM
SDS
2 % (w/v)
FACS Wash Buffer
PBS + 1%FCS + 1% NaN3
FACS Fixative Solution
PBS + 2% formaldehyde solution
Phosphate-Buffered Saline (PBS)
NaCl
137 mM
KCl
2.7 mM
Na2HPO4
10 mM
KH2PO4
2 mM
The above components were dissoolved in water and adjusted pH to 7.4 with HCl.
Tris-HCl (1M)
121.1g of Tris base was dissolved in 800ml of water. The pH was adjusted to the desired
10X Tris EDTA (TE)
pH 7.4
100 mM Tris-Cl (pH 7.4)
10 mM EDTA (pH 8.0)
pH 7.6
100 mM Tris-Cl (pH 7.6)
10 mM EDTA (pH 8.0)
pH 8.0
100 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
SDS (20% w/v)
200 g of electrophoresis-grade SDS was dissolved in 900 ml of water. The mixture was
heated to 68°C and stirred with a magnetic stirrer to assist dissolution. The pH was adjusted to
7.2 by adding a few drops of concentrated HCl. The volume was adjusted to 1 liter with
water.
EDTA (0.5 M, pH 8.0)
186.1 g of disodium EDTA was added to 800 ml of H2O. The mixture was stired vigorously
on a magnetic stirrer. The pH was adjusted to 8.0 with NaOH. The disodium salt of EDTA did
not go into solution until the pH of the solution was adjusted to ~ 8.0 by the addition of
NaOH.
 
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Materials
49
Solutions for plasmid minipreparation
Resuspension solution (Sol I)
Glucose
50 mM
Tris-HCl pH 8.0
25 mM
EDTA pH 8.0
10 mM
Autoclaved and stored the solution at 4°C
Alkaline lysis solution (Sol II)
NaOH
0.2 M
SDS
1 % (w/v)
Sol II was prepared fresh and used at room temperature
Neutralization solution (Sol III)
5 M Potassium acetate
60 ml
Glacial acetic acid
11.5 ml
ddH2O 28.5 ml
The solution was stored at 4°C and transferred to ice before use
0.1% Diethylpyrocarbonate (DEPC)
1 g DEPC was dissolved in 1 L water and mixed it vigiously. It let stand with loose lid
overnight at room temperature or at 37°C for 1h and autoclaved for 15 minutes at 15 psi on
liquid cycle. DEPC cannot be used to treat Tris buffer.
2.3.7 Plasmids and vectors
pCDNA 3.1 vector
Invitrogen GmbH, Karlsruhe, Germany
pEntr1A
Invitrogen GmbH, Karlsruhe, Germany
PINCO
Retroviral hybrid vector with LTR derived from Moloney
murine Leukemia virus. The main characteristics of this vector
is the presence of the EBV origin of replication and the EBNA-
1 gene and the presence of the cDNAs that encodes for the
EGFP, controlled by a cytomegalovirus promoter.This vector
allows high-efficiency gene transfer (Grignani et al., 1998).
PAULO
This vector is a modified pinco vector in which GFP is replaced
by LNGFR
PIDE
This vector is based on pinco vector in which GFP is deleted and
Hind III site can be used for cloning gene instead of GFP
Pinco∆CMV
Retroviral pinco based vector without any reporter gene
 
Page 50
Materials
50
pPRIBA2
IBA, Gottingen , Germany
pGEX4T3
Amersham Biosciences, Freiburg, Germany
2.3.8 Bacterial E.Coli Strain and genotype
E. coli – HB101, BL21, JM83, DB 3.1,
Invitrogen, Karlsruhe, Germany
2.3.9 Medium for bacterium
LB medium
Bacto-Tryptone
1 % (w/v)
Bacto-Yeast-Extrac
0.5 % (w/v)
NaCl
1.5 % (w/v)
Adjuted pH to 7.4 with NaOH and autoclaved
LB agar plates
Bacto-Agar
1.5 % (w/v)
Bacterium freezing solution
Glycerin
65 % (v/v)
MgSO4
0.1 M
TrisHCl, pH 8.0
0.025M
SOC
Invitrogen, Karlsruhe, Germany
2.3.10 Cell lines
2.3.10.1 Ph+ cells
CML blast cell lines
In CML cells, the translocation product encodes for p210
BCR/ABL
.
BV173: Human B cell precursor leukaemia cell line, established from the peripheral blood of
a patient with chronic myeloid leukaemia (CML) in lymphoid blast crisis. Contains the t(9;22)
b2-a2 fusion gene. Obtained from DSMZ, Germany
K562: Established from the pleural effusion of a patient with chronic myeloid leukaemia
(CML) in myeloid blast crisis. Cells carry the Ph chromosome with the Bcr-Abl b3-a2 fusion
gene. Obtained from DSMZ, Germany
Ph+ ALL
In Ph+ cells, the translocation product encodes for p185
BCR/ABL
.
 
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Materials
51
Sup-B15: Human B cell precursor leukemia cell line, established from the bone marrow of a
patient with acute lymphoblastic leukemia, carrying the ALL-variant (m-bcr) of the
BCR/ABL fusion gene (e1-a2). Obtained from DSMZ, Germany
Tom-1: human B cell precursor leukemia. Established from the bone marrow of a patient with
refractory Ph chromosome acute lymphoblastic leukemia (ALL); described to carry the ALL-
variant (m-BCR) of the BCR/ABL fusion gene (e1-a2) obtained from DSMZ, Germany.
2.3.10.2 Other Cell lines
Nalm-6: Human lymphoblastic B cell line; Ph-
Ba/F3: IL-3 dependent murine pro B cell line established from peripheral blood; apparently
derived from BALB/c mouse.
32D: IL-3 dependent murine myeloid cell line established from peripheral blood.
Rat-1: Rat fibroblast cells line
293T: Transformed human embryonal kidney cell line
Phoenix: Based on the 293T cell line (transformed human embryonal kidney cell line).
Expresses the retroviral structural genes gag, pol und env. It is also known as a packaging cell
line. The ecotropic packaging cell line delivers genes only to dividing murine or rat cells.
All cells were obtained from the “Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH” (DSMZ) (German Collection of Microorganisms and Cell Cultures),
with the exception of the ecotrophic Phoenix cells which were obtained from Nolan lab,
Standford, USA.
2.3.11 Medium for Cell culture
L-glutamine
1 % (v/v)
FBS
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v)
Added to DMEM and RPMI medium for adherent and suspension cells respectively
Medium for 32D/Baf3 cells
L-glutamine
1 % (v/v)
FBS
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v)
mIL-3
10 ng /ml
Added the above to RPMI medium
Medium for Nalm-6, BV-173 and K562
L-glutamine
1 % (v/v)
 
Page 52
Materials
52
FBS
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v
Added the above to RPMI medium
Medium for Tom-1
L-glutamine
1 % (v/v)
FBS
20 % (v/v)
Penicillin/Streptomycin
1 % (v/v
Added the above to RPMI medium
Medium for Sup-B15
L-glutamine
1 % (v/v)
FBS
20 % (v/v)
Penicillin/Streptomycin
1 % (v/v
Added the above to Mycosa medium
Medium for Sca1+/lin- mouse BM cells
L-glutamine
1 % (v/v)
FBS(Hyclon)
10 % (v/v)
Penicillin/Streptomycin
1 % (v/v)
mIL-3
20 ng /ml
mIL-6
20 ng /ml
mSCF
100 ng /ml
Added the above to ISCOVE medium
Cell freezing medium
Solution I
RPMI/DMEM
70 % (v/v)
FBS
30 % (v/v)
Solution II
RPMI/DMEM
80 % (v/v)
DMSO
20 % (v/v)
2.3.12 Materials for animal experiments
2.3.12.1 Mice
The C57BL/6N mice (age between 6-8 weeks, all female) were purchased from Charles River
Laboratories GmbH in Munich, Germany and served as the recipient mice for all the animal
experiments.
 
Page 53
Materials
53
2.4 Miscellaneous
Nucleospin
R
Mini plasmid DNA isolation Kit
Macherey-Nagel, Dueren Germany
Nucleospin
R
Maxi plasmid isolation kit
Macherey-Nagel, Dueren Germany
Gel cleaning kit
Macherey-Nagel, Dueren Germany
PCR cleaning kit
Macherey-Nagel, Dueren Germany
Qiagen gel extraction Kit
Qiagen, Duesseldorf, Germany
Qiagen Plasmid kit Mini, Midi and Maxi
Qiagen, Duesseldorf, Germany
Qiagen PCR purification Kit
Qiagen, Duesseldorf, Germany
Quick change site mutagenesis kit
Stratagene, La Jolla, USA
Endo Toxin removal kit
Profos AG, Regensburg, Germany
Cell proliferation (XTT) kit
Amersham/Pharmacia Biotech, Freiburg,
Germany
Protein concentrator (Vivapore)
VivaScience, Hannover, Germany
 
Page 54
Methods
54
3 Methods
3.1 Preparation of plasmid DNA
3.1.1 Transformation of E.coli
A frozen vial of competent E.coli bacteria was thawed on ice. The transforming DNA (up to
25ng per 50ul competent bacterium or 10ul ligation product) was pipetted directly to
competent E.coli bacteria and mixed by swirling the tubes gently several time. 

Laboratoreum Leveranciers

ABCell
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Amresco Inc.
AMS
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Atlantic Pharmaceuticals, Inc.
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Celltech Group
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Charles River Laboratories, Inc.
CHEMICON
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CuraGen Corporation

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Eppendorf
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FAAR Biotechnology Group, Inc.
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Genmab
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Lab Vision
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Lee BioMolecular Research Laboratories, Inc.
Leica
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MED Systems
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MWG
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NEN Life Science Products
Nest Group, Inc.
NEUROLAB
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Novagen
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PanVera Corporation
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PharMingen
Pierce Biotechnology, Inc.
Pineda – Antikörper-Service
Polyfiltronics, Inc.
Premier Biosoft International
Princeton Separations, Inc.
Promega Corporation
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QED Bioscience
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R & D Systems
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responsif gmbh
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Roche Applied Science
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Santa Cruz Biotechnology, Inc.
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Sequana Therapuetics, Inc.
Serotec Antibodies
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Sigma Chemical Co.
Sigma-Aldrich
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SPI Supplies
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Steraloids
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T&t Research
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Telluride Pharmaceutical Corporation
Terumo
Textco, Inc.
The Franklin Search Group, Inc.
The Lovelace Institutes
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Thetagen
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Toronto Research Chemicals, Inc.
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Transgenics in Berlin-Buch GmbH
Trevigen, Inc.

USBiological
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VaccineLab
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Vyker Instruments
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Washington Biotechnology Inc.
Whatman Labsales
Wilex
wRight BioDiagnostics, L.C.

Xenogen Transgenic Technologies
XOMA

ZYMED Laboratories,Inc.
Zymed Medical Instrumentation

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Aat Bioquest, Inc. www.aatbio.com
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Accegen Biotech www.accegen.com
Accurate Chemical & Scientific Co. www.accuratechemical.com
Acrobiosystems Usa www.acrobiosystems.com
Adams & Chittenden Scientific Glass www.adamschittenden.com
Advanced Targeting Systems atsbio.com
Affinity Biologicals Inc. www.affinitybiologicals.com
Affinity Immuno Inc. affinityimmuno.com
Agrenvec www.agrenvec.com
Aic Biotech www.aicbiotech.com
Aldevron Freiburg Gmbh www.aldevron.com
Alomone Labs www.alomone.com
Alpco www.alpco.com
American Type Culture Collection www.atcc.org
Ampersand Biosciences www.ampersandbio.com
Aniara www.aniara.com
Ansh Labs www.anshlabs.com
Antibodies Inc. www.antibodiesinc.com
Antibodies-Online Gmbh www.antibodies-online.com
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Arbor Assays, Inc. www.arborassays.com
Arista Biologicals Inc. www.aristabiologicalsinc.com
Astarte Biologics, Llc. www.astartebio.com
Aves Labs www.aveslab.com
Binding Site Immunologicals Group www.immunologicals.com
Bio X Cell www.bxcell.com
Biocytex www.biocytex.fr
Biofront Technologies www.biofronttech.com
Biogenes Gmbh www.biogenes.de
Biolegend www.biolegend.com
Biomatik www.biomatik.com
Biomolecular Discovery Services Llc www.biomoldiscserv.com
Biorbyt Ltd. www.biorbyt.com
Biosearch Technologies www.biosearchtech.com
Biospacific Inc. www.biospacific.com
Bioss Antibodies www.biossusa.com
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Boster Bio www.bosterbio.com
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Cooperative Human Tissue Network www.chtn.org
Coriell Inst. For Medical Research www.coriell.org
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Dendritics Sas www.dendritics.net
Deutsche Sammlung Von Mikroorganismen www.dsmz.de
Developmental Studies Hybridoma Bank dshb.biology.uiowa.edu
Dianova Gmbh www.dianova.com
Drg International Inc. www.drg-international.com
Dsmz Gmbh www.dsmz.de
Eagle Biosciences Inc. www.eaglebio.com
Ecm Biosciences Llc www.ecmbio.com
Ellegaard Goettingen Minipigs Awww.minipigs.dk
Enquire Bioreagents www.enquirebio.com
Enzyme Research Laboratories Inc. www.enzymeresearch.com
Euromabnet www.euromabnet.com
European Collection Of Cell Cultures (Ecacc) www.phe-culturecollections.org.uk
European Directorate For The Quality Of Medicines & Healthcare www.edqm.eu
European Malaria Reagent Repository www.malariaresearch.eu
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Finetest www.fn-test.com
Fujifilm Wako Chemicals Usa Corp. www.wakousa.com
Genetex Inc. www.genetex.com
Haematologic Technologies Inc. www.haemtech.com
Hemacare Corporation www.hemacare.com
Huabio Inc. www.huabio.com
Hycult Biotech www.hycultbiotech.com
Hytest Ltd. www.hytest.fi
Ibt Bioservices www.ibtbioservices.com
Immundiagnostik Ag www.immundiagnostik.com
Immunodiagnostic Systems www.idsplc.com
Immunoglobe Antikorpertechnik Gmbh immunoglobe.com
Immunological & Biochemical Testsystems Gmbh www.ibtsystems.de
Immunostar Inc. www.immunostar.com
Immuquest Ltd. www.immuquest.com
International Blood Group Reference Lab ibgrl.blood.co.uk
Jackson Immunoresearch Labs Inc. www.jacksonimmuno.com
Kerafast, Inc. www.kerafast.com
Kronus, Inc. www.kronus.com
Leading Biology Inc. www.leadingbiology.com
Life Diagnostics Inc. www.lifediagnostics.com
Lifespan Biosciences Inc. www.lsbio.com
Mabtech Ab www.mabtech.com
Medgene Labs www.medgenelabs.com
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Miami Serpentarium Laboratories www.miamiserpentarium.com
Miltenyi Biotec B.V. & Co. Kg www.miltenyibiotec.com
Molecular Depot moleculardepot.com
Mybiosource Inc. www.mybiosource.com
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Nat’L. Institute For Biological www.nibsc.org
Nci Biological Resources Branch web.ncifcrf.gov
Neuromab www.antibodiesinc.com
Nih Aids Reagent Program www.aidsreagent.org
Nih Nat’L. Institute On Aging www.nia.nih.gov
Nittobo America Inc. www.nittobous.com
Nkmax Co. Ltd. (Formerly Atgen Co. Ltd.) www.nkmaxbio.com
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Origene Technologies www.origene.com
Oz Biosciences Sas www.ozbiosciences.com
Pel-Freez Biologicals www.pelfreez-bio.com
Peninsula Laboratories International, Inc. www.penlabs.com
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Polymun Scientific Gmbh www.polymun.com
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Protein Mods www.proteinmods.com
Protos Immunoresearch www.protosimmuno.com
Qed Bioscience Inc. www.qedbio.com
Rapid Novor Inc. www.rapidnovor.com
Roche www.sigmaaldrich.com
Rockland Immunochemicals Inc. rockland-inc.com
Rti, Llc www.4rtilab.com
Scripps Laboratories www.scrippslabs.com
Sicgen Antibodies www.sicgen.pt
Sino Biological, Inc. www.sinobiological.com
Sjk Global Llc sjkglobalkc.com
Southernbiotech www.southernbiotech.com
St John’S Laboratory www.stjohnslabs.com
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Synaptic Systems Gmbh www.sysy.com
Titermax www.titermax.com
United States Biological www.usbio.net
University Of California Davis 
Virostat Inc. www.virostat-inc.com
Vmrd Inc. www.vmrd.com
Wsu Monoclonal Antibody Center www.vetmed.wsu.edu
Zebrafish International Resource Center (Zirc) zebrafish.org

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