Protein Technologies

  • We provide expertise in recombinant protein technologies and genome engineering.
  • We provide expertise in recombinant protein technologies and genome engineering.
  • We provide expertise in recombinant protein technologies and genome engineering.
  • We provide expertise in recombinant protein technologies and genome engineering.

The mission of the Protein Technologies Facility (ProTech) is to further research in molecular and cell biology, protein biochemistry, and structural biology by overcoming major bottlenecks in these fields.

Our core services include molecular cloning, protein production and purification, and biophysical characterization of proteins. We also offer services surrounding the CRISPR/Cas9 genome engineering technology.

If you are interested in our services and would like additional information or have specific project questions...
GET IN TOUCH WITH US!

Services

    Molecular Cloning and CRISPR Reagents

    Molecular Cloning and CRISPR Reagents

    We offer molecular cloning services as a stand-alone service or as the preliminary step in protein production services. We can also generate DNA, RNA, and protein reagents appropriate for your CRISPR/Cas9 experiment.

    Molecular Cloning

    • Sequence and Ligation Independent Cloning (SLIC) into E.coli, insect cell, or mammalian expression vectors
    • Cloning of multi-gene expression constructs
    • Site-directed mutagenesis

    A full list of vectors is available under the Resources section.

    For advice on your cloning/expression project please contact Peggy Stolt-Bergner to discuss your project.

    CRISPR Reagents

    • Design of gRNAs
    • Preparation of DNA constructs containing gRNA(s) and Cas9
    • Preparation of RNA
    • Preparation of purified Cas9 protein

    For help with experimental design please contact Krzysztof Chylinski.

    Protein Production and Purification

    Protein Production and Purification

    We offer protein production in E. coli or the baculovirus system either as a stand-alone service or in combination with protein purification. Plasmids must be IPTG-compatible (E. coli) or compatible with Tn7 transposition (BEVS).

    For advice on which expression system is appropriate for your protein of interest please contact Peggy Stolt-Bergner to discus your project.

    To request service, please download and fill out the appropriate request form (see How to Order Service).

    E. coli expression services

    We perform E. coli expression in auto-induction media using a variety of different BL21-based strains. You can find a description of available strains in the Resources section. Optimization can be performed by screening different expression strains or different expression temperatures.

    We offer:

    • Small-scale (2ml) expression and purification test
    • Expression optimization (2ml scale)
    • Test-scale expression
    • Large-scale expression
    • Fermentation (through Eucodis Bioscience)

    BEVS expression services

    The Baculovirus Expression Vector System is a eukaryotic expression system that can often be successfully used to generate milligram amounts of more complex and/or post-translationally modified eukaryotic proteins.

    ProTech employs the MultiBacTM system, originally developed by Imre Berger (EMBL-Grenoble), that features:

    • Viral gene deletions to improve protein expression
    • Facilitated expression of protein complexes from one virus
    • Ease of monitoring protein expression via a virally encoded YFP marker protein

    Protech has licensed this system from Geneva Biotech (www.geneva-biotech.com). We offer:

    • Start-up service:  initial virus generation including a small-scale (50 ml) expression test in three expression conditions
    • Expression optimization (50 ml scale)
    • Virus amplification
    • Generation of Baculovirus Infected Insect Cells (BIICs)
    • Large-scale expression

    Purification services

    As protein purification can be quite difficult and time-consuming our services are focused on small-scale testing to identify and optimize a purification protocol for a particular target protein or protein complex. Large-scale purification is then carried out by the user in collaboration with ProTech. In exceptional cases ProTech can also perform large-scale purification.

    We also offer standard laboratory reagents and enzymes. A complete list is available in the Resources section.

    We offer:

    • Small-scale (2 ml) expression and purification (E. coli)
    • Test-scale (50 ml) purification (insect cells)
    • Purification optimization
    • Training/access to Äkta Purifier systems
    • Full purification service (exceptional cases)
    • Protein reagents (enzymes, growth factors, etc)

    Protein Characterization

    Protein Characterization

    We offer protein characterization services using a variety of biophysical techniques to study protein stability, protein oligomeric state, protein secondary structure, or to analyze biomolecular interactions.

    We also provide instrument training and access for some of these methods.

    Often many different biophysical techniques could be applied to answer a specific question. To gain an overview of what techniques are available and which one applies best to your project please contact
    Peggy Stolt-Bergner or Arthur Sedivy.

    To request service please see How to Order Service.

    Circular Dichroism (CD)

    Circular Dichroism is a spectroscopic technique that uses circularly polarized light to study the structure of chiral molecules, such as proteins.

    The CD spectrum of a protein can be used to determine protein secondary structure or some aspects of tertiary structure.  CD can also be used to study protein stability using thermal melt analysis to determine protein melting temperatures (Tm’s). We perform CD on a Chirascan Plus CD spectrometer from Applied Photophysics.  Experiments can be carried out for you, or alternatively you may receive training and can then book the instrument yourself.

    Dynamic Light Scattering (DLS)

    DLS is a label free technique, mainly used for protein quality control, as one can detect size distributions of molecules and therefore aggregation and oligomerization of your protein of interest. In contrast to SEC-MALLS no size exclusion column is needed, therefore also “sticky” samples can be analyzed in a few minutes.

     

     

    Differential Scanning Calorimetry (DSC)

    DSC is a label free technique that can be used to determine thermodynamic parameters of your system, for instance protein folding energies (enthalpies and entropies) as well as melting temperatures. Thermodynamic differences of mutants or in the presence of ligands can be measured to evaluate stabilizing/destabilizing effects. Other phase changes (e.q. lipid phase changes, RNA structural changes) can be observed with this technique as well.

     

     

    Differential Scanning Fluorimetry (DSF) or Thermofluor

    Thermofluor is used to conduct up to 96 thermal shift assays in parallel to gain insight into conditions that enhance protein stability. Protein samples are mixed and heated in the presence of a dye that fluoresces only when exposed to a hydrophobic environment. The fluorescence increase upon temperature-dependent protein unfolding can be used to determine the melting temperature of the protein sample.

    Screening protein thermal stabilities under different buffer conditions or in the presence of different ligands will reveal optimal conditions for further experiments (structural analysis, thermodynamic properties, etc).

     

     

    Microscale Thermophoresis (MST)

    MST is a novel technique used to determine molecular affinities. Thermophoretic changes upon molecular interaction can be monitored by fluorescent labeling or via tryptophan fluorescence, revealing binding constants in the range of nM to mM.  Unlike many other methods, MST experiments can be performed rapidly and with small amounts of sample. 

    We have two instruments for performing MST experiments, the NT.115 and the NT.LabelFree (see Resources section).  MST experiments can be performed as a service, or you can receive training and then book the instruments yourself.

    SEC-MALLS

    Analytical Size Exclusion Chromatography and Multi Angle Laser Light Scattering (SEC-MALLS) can be used to assess the oligomeric state and homogeneity of protein samples in solution. In addition one can very accurately determine molecular weight of proteins / protein complexes and protein concentrations (UV280 independent). This method is often used as a method of protein quality control to determine whether purified protein samples are homogeneous and monomeric.

    We perform aSEC on an Aekta micro system using either a Superdex75 (molecular weight range from 3-70 kDa) or Superdex 200 column (molecular weight range from 10-600 kDa).

     

     

    Simple Western - Size Separation

    Simple Western - Automated SDS-PAGE and immunodetection

    The new Simple Western (SW) technology performs separation of protein samples based on either size or charge, combined with immunodetection.

    Size separation via Simple Western performs SDS-PAGE and Western blotting in an automated format by combining capillary electrophoresis with immunodetection. Simple Western size separation provides sensitivity equal to that of a Western blot and additionally allows for quantifiable, reproducible data and consumes low amounts of sample and primary antibody.

    For more information, please contact Peggy Stolt-Bergner.

    See examples of Simple Western size separation.

    Simple Western - Charge Separation

    Simple Western - Automated isoelectric focusing and immunodetection

    The new Simple Western (SW) technology performs separation of protein samples based on either size or charge, combined with immunodetection.

    Charge separation performs isoelectric focusing of native protein samples in an automated format, also using capillary electrophoresis combined with immunodetection.  Simple Western charge separation provides extremely high sensitivity and allows for quantifiable identification of protein isoforms in a sample (for example, identification of different phospho-isoforms).

    For more information, please contact Peggy Stolt-Bergner.

    CRISPR Lab

    CRISPR Lab

    The mission of CRISPR-Lab is to help VBC scientists in the optimal utilization of CRISPR/Cas9 technology by providing both knowledge and necessary customized materials.

    We are open to collaborations on specific research goals/projects. If you are interested in using the facility or have any questions please contact Krzysztof Chylinski.

    Please refer to our services and ongoing projects below. CRISPR/Cas9 services are offered for academic use only.

    CRISPR-Lab has been funded by contributions from MFPL, GMI, IMP, and IMBA, as well as a generous private donation from Prof. Dr. Renée Schroeder.

    Basic CRISPR/Cas9 services

    Our services include:

    • Introduction to the CRISPR/Cas9 technology and help with experimental design
    • Design of guide RNAs
    • Communication platform and repository of the CRISPR/Cas9-related materials (a VBC-wide CRISPR Wiki page will be available soon)
    • Basic reagents (please see “Molecular Cloning and CRISPR Reagents”)

    CRISPR/Cas9 in A. thaliana

    CRISPR-Lab has established the CRISPR/Cas9 technology in A. thaliana, through a project funded jointly by CSF-ProTech and CSF-PlantS together with 10 labs from GMI, MFPL, BOKU and ISTA.

    We provide the following A. thaliana services for gene knock-out projects:

    • Project design and generation of a binary vector containing Cas9 and guide RNAs
    • Above service plus transformation of A. thaliana with the desired construct (we provide unselected T1 seeds)
    • Above services plus selection (we provide T2 seeds from 10+ selected T1 plants)
    • Above services plus characterization of T2 plants (we provide T3 seeds with a verified frameshift mutation (indel))

    For knock-in projects please contact the facility.

    CRISPR/Cas9 in mammalian systems

    CRISPR-Lab can provide support with establishment of CRISPR/Cas9 in a variety of mammalian cell lines through a collaboration project. Please contact Krzysztof Chylinski if you are interested in such a project.

    Example projects:

    • Establishment of CRISPR/Cas9 knock-in technology in mouse embryonic fibroblasts (MEFs), together with the lab of Prof. Dr. Manuela Baccarini.
    • Establishment of CRISPR/Cas9 knock-out technology in several cell lines

    Pictures coming soon!

    CRISPR/Cas9 Research and Development

    CRISPR-Lab also works on CRISPR/Cas9 research and development projects. Most of this work is funded by a grant from the BMWFW awarded to CSF and our partner Eucodis Bioscience.

    Projects include:

    • Characterization of Cas9 orthologues
    • Optimization of the CRISPR/Cas9 system to determine best practices
    • Applications of CRISPR/Cas9 to recombinant protein production

    Pictures coming soon!

    Resources

    Antibodies

    Primary antibodies available at the ProTech for Simple Western size separation analysis.  Supplier and catalog number are indicated in parentheses :

    • anti-His (Qiagen, 34460)
    • anti-Strep (IBA, 2-1507-001)
    • anti-GST  (GE Healthcare, RPN1236)
    • anti-GFP/anti-YFP (Cell Signaling, 2555)
    • anti-Flag (Sigma, F1804)
    • anti-Myc (Cell Signaling, 2278)

    Instruments

    The following equipment is available for booking after appropriate training:

    Äkta Purifier
    An Äkta Purifier FPLC system for protein purification. Users must provide their own columns.

    Monolith NT.115 BLUE/RED
    An instrument for Microscale Thermophoresis (MST) measurements using fluorescence detection with either a blue (470/520) or red (625/680)filter.

    Monolith NT.LabelFree
    An instrument for Microscale Thermophoresis (MST) measurements using detection via native tryptophan fluorescence.

    Chirascan Plus CD Spectrometer
    Instrument for Circular Dichroism measurements, including far-UV spectra, near-UV spectra, and thermal melting curves.

    Protein reagents

    Coming soon...

    E. coli strains

    We have the following E. coli expression strains available for expression screening:

    • BL21(DE3)
    • LEMO(DE3)
    • Rosetta2(DE3)
    • Rosetta-gami(DE3)
    • LOBSTR(DE3)

    E. coli and BEVS vectors

    E. coli and BEVS vectors with the following N-terminal tags followed by a 3C protease cleavage site

    • His6
    • His10
    • Trx-His6
    • His6-GST
    • His6-MBP
    • His6-SUMO (E. coli only!)
    • His6-mEGFP
    • His6-VenusYFP
    • TwinStrep
    • c-Myc
    • FLAG

    E. coli and BEVS vectors with the following C-terminal tags

    • StrepII
    • His6
    • mEGFP-His6
    • VenusYFP-His6
    • Honey bee mellitin secretion signal, C-terminal His6 (BEVS only)

    E. coli and BEVS vectors with the following N and C-terminal tags

    • N-terminal His6-3C protease site, C-terminal StrepII
    • N-terminal TwinStrep-3C protease site, C-terminal His6

    Mammalian expression vectors based on the pIRES vector

    N-terminal:

    • His6-mGFP
    • His6-VenusYFP
    • His6-mCherry
    • His6-FusionRED

    C-terminal:

    • mGFP-His8
    • VenusYFP-His6
    • mCherry-His6
    • FusionRED-His6

    Resources available at Partner Institutions

    User Information

    How to order service

    If you are a new user, please contact Peggy Stolt-Bergner before placing an order to discuss your project requirements.

    To order service, users must fill out a Project Agreement form.  It is not possible for us to process service requests without a Project Agreement form. Project Agreement forms can be obtained from Peggy Stolt-Bergner or other ProTech staff members.

    VBC members (IMP, IMBA, GMI, MFPL) can log in to MyVBCF with their existing institute user name and password.  Non-VBC members can request a MyVBCF account (see the MyVBCF section of the website).

    For questions, please contact protech@vbcf.ac.at.

     

     

    Training

    We also offer training and instrument access for Circular Dichroism and Microscale Thermophoresis. In addition we offer training and booking on one of our Äkta Purifier systems. To schedule training please contact Arthur Sedivy or Peggy Stolt-Bergner.

    The instrument booking system can be found here.

    Facility usage policy

    The ProTech Facility Usage Policy can be downloaded here. It is assumed that users who order service have read and understood this policy as well as the VBCF General Cooperation Conditions.

    For questions please contact Peggy Stolt-Bergner.

    Citing the facility

    Continuation of funding for VBCF-ProTech (and all other VBCF facilities) by the City of Vienna and the Austrian Federal Ministry of Science, Research, and Economy is dependent on documented evidence of contributions to scientific output.

    Therefore it is absolutely required to acknowledge use of the facility when publishing work in which ProTech services were utilized. Please also acknowledge training and instrument access provided by ProTech.

    A simple statement is sufficient and can either be placed in the Materials and Methods section or in the Acknowledgements.

    Suggested format:
    "ProTech service" was performed by the VBCF Protein Technologies Facility (www.vbcf.ac.at).
    Your publication will then be listed on our website.

    Publications

    Genetic code expansion for multiprotein complex engineering

    Koehler C, Sauter PF, Wawryszyn M, Girona GE, Gupta K, Landry JJ, Fritz MH, Radic K, Hoffmann JE, Chen ZA, Zou J, Tan PS, Galik B, Junttila S, Stolt-Bergner P, Pruneri G, Gyenesei A, Schultz C, Biskup MB, Besir H, Benes V, Rappsilber J, Jechlinger M, Korbel JO, Berger I, Braese S, Lemke EA. (2016).

    The Sodium Glucose Cotransporter SGLT1 is an Extremely Efficient Facilitator of Passive Water Transport
    Erokhova L, Horner A, Ollinger N, Siligan C, Pohl P. (2016).
    J Biol Chem epub March 4, 2016 (abstract)

    The structure and regulation of human muscle alpha-actinin
    Ribeiro EA Jr, Pinotsis N, Ghisleni A, Salmazo A, Konarev PV, Kostan J, Sjöblom B, Schreiner C, Polyansky AA, Gkougkoulia EA, Holt MR, Aachmann FL, Zagrović B, Bordignon E, Pirker KF, Svergun DI, Gautel M, Djinović-Carugo K. (2014).
    Cell 159(6):1447-60 (abstract)

    Structure-Function Analysis of Heterodimer Formation, Oligomerization and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH.
    Badarau A, Rouha H, Malafa S, Logan DT, Håkansson M, Stulik L, Dolezilkova I, Teubenbacher A, Gross K, Maierhofer B, Weber S, Jägerhofer M, Hoffmann D, Nagy E. (2014).
    J Biol Chem epub 2014 Nov 3 (abstract)

    Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein
    Vidilaseris K, Shimanovskaya E, Esson HJ, Morriswood B, Dong G (2014).
    J Biol Chem 289(34):23870-81 (abstract)

    A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element
    Herzog VA, Lempradl A, Trupke J, Okulski H, Altmutter C, Ruge F, Boidol B, Kubicek S, Schmauss G, Aumayr A, Ruf M, Pospisilik A, Dimond A, Senergin HB, Vargas ML, Simon JA, Ringrose L. (2014).
    Nat Genet 46(9):973-981 (abstract)

    The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium.
    Santos JA, Alonso-García N, Macedo-Ribeiro S, Pereira PJ. (2014).
    Proc Natl Acad Sci 111(22):E2251-60 (abstract)

    Characterization and Structure of the Vaccinia Virus NF-kB Antangonist A46.
    Fedosyuk, S., Grishkovskaya I., de Almeida Ribeiro E Jr., Skern T. (2014).
    J Biol Chem 289(6):3749-62 (abstract)

    Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire.
    Medvedovic J, Ebert A, Tagoh H, Tamir IM, Schwickert TA, Novatchkova M, Sun Q, Huis In't Veld PJ, Guo C, Yoon HS, Denizot Y, Holwerda SJ, de Laat W, Cogne M, Shi Y, Alt FW, Busslinger M (2013).
    Immunity 39(2):229-44 (abstract)

    Sufficient amounts of functional HOP2/MND1 complex promote interhomolog DNA repair but are dispensable for intersister DNA repair during meiosis in Arabidopsis.
    Uanschou C, Roncerat A, Von Harder M, De Muyt A, Vezon D, Pereira L, Chelysheva L, Kobayashi W, Kurumizaka H, Schloegelhofer P, Grelon M (2013).
    Plant Cell 25(12):4924-40 (abstract)

    Team

    Peggy Stolt-Bergner

    Peggy Stolt-Bergner

    Peggy Stolt-Bergner is the head of ProTech and provides advice and expertise on projects in all service areas

    Core Facility Head
    VBC2 / 1OG-07

    Christian Aigner

    Christian Aigner

    Laboratory Assistant
    VBC2 / 1OG-06

    Ivana Bilusic-Vilagos

    Ivana Bilusic-Vilagos

    Laboratory Scientist
    VBC2 / 1OG-05

    Monika Borowska

    Monika Borowska

    Monika Borowska works within the Laura Bassi Centre for Optimized Structural Studies, where CSF is a partner

    Laboratory Scientist
    VBC2 / 1OG-05

    Krzysztof Chylinski

    Krzysztof Chylinski

    Chris Chylinski provides advice and expertise on use of the CRISPR/ Cas9 system through the CRISPR-Lab initiative

    Staff Scientist
    VBC2 / 1OG-05

    Anita Lehner

    Anita Lehner

    Anita Lehner is responsible for the BEVS expression and protein purification services

    Laboratory Scientist
    VBC2 / 1OG-06

    Jana Neuhold

    Jana Neuhold

    Jana Neuhold is responsible for the molecular cloning, CRISPR reagents, and E. coli expression services

    Laboratory Scientist
    VBC2 / 1OG-06

    Katharina Radakovics

    Katharina Radakovics

    Laboratory Scientist
    VBC2 / 1OG-06

    Vera Karolina Schoft

    Vera Karolina Schoft

    Staff Scientist
    VBC2 / 1OG-06

    Arthur Sedivy

    Arthur Sedivy

    Arthur Sedivy is responsible for the biophysical characterization services

    Laboratory Scientist
    VBC2 / 1OG-07

    Contact

    List of services

    E. coli Expression Screening
    Time required: 1 week

    • Transformation into standard ProTech cell line
    • Set-up of small-scale (2 mL) cultures
    • Small-scale affinity purification
    • Expression Analysis via SDS-PAGE

     

    E. coli Expression Optimization
    Time required: 2 weeks
    Testing expression of 1 construct under different conditions: 3 cell lines, 2 media, 2 temperatures

    • Transformation
    • Set-up of small-scale (2 mL) cultures
    • Small-scale affinity purification
    • Expression Analysis via SDS-PAGE

     

    Test-scale E. coli Expression
    50 mL scale expression to be combined with purification test

    • Transformation
    • Set-up of 50 mL culture
    • harvesting of cells

     

    Large-scale E. coli Expression

    • Transformation
    • Set-up of culture
    • harvesting of cells

     

    Fermentation
    5, 10, 50 L volumes or 4 x 1 L optimization trials available through our partner Eucodis Bioscience. Please contact ProTech for details.

    Analytical Size Exclusion Chromatography (aSEC)

    aSEC can be used to assess the oligomeric state and homogeneity of protein samples in solution. This method is often used as a method of protein quality control to determine whether purified protein samples are homogeneous and monomeric.

    We perform aSEC on an Aekta micro system using either a Superdex75 (molecular weight range from 3-70 kDa) or Superdex 200 column (molecular weight range from 10-600 kDa).

    Circular Dichroism

    CD can also be used to study protein stability using thermal melt analysis to determine protein melting temperatures (Tm’s). Such experiments can be used to compare the stability of different protein mutants or to compare the stability under different buffer conditions or in the presence of ligands.

    We perform CD on a Chirascan Plus CD spectrometer from Applied Photophysics. Experiments can be carried out for you, or alternatively you may receive training and can then book the instrument yourself.

    The CD spectrum of a protein collected in the far-UV wavelength range (180 – 260 nm) reveals details of the secondary structure content. This information can be used to study the influence of point mutations on protein structure, to compare structural changes in the presence of ligands, or to ensure reproducibility and consistency between different protein batches, for example.

    CD spectra in the near-UV wavelength range (230-350 nm) can also be used to study tertiary structural changes due to changes in the aromatic residues Tryptophan and Tyrosine in this wavelength range.

    In addition CD can be used to study protein stability using thermal melt analysis to determine protein melting temperatures (Tm’s). Such experiments can be used to compare the stability of different protein mutants or to compare the stability of the same protein under different buffer conditions or in the presence of different ligands.

    All of our CD data is collected on our Chirascan Plus CD spectrometer from Applied Photophysics. Experiments can be carried out for you, or alternatively you may receive training and can then book the instrument yourself.

    Start-up package

    Service includes steps 1-3 for one construct and storage of initial viruses for 1 year. The user must either order molecular cloning service or provide the transfer plasmid.

    Time required: 2-3 weeks

    1. Generation of bacmid:

      • ransformation of the transfer plasmid into E. coli containing the EMBacY bacmid
      • Selection for transposition into the bacmid via blue/white screening
      • Purification of bacmid DNA

    2. Generation of recombinant baculovirus:

      • Transfection of bacmids into insect cells
      • Harvesting of initial virus-containing supernatants
      • Sterile-filtration and storage of initial viruses

    3. Qualitative expression test:

      • Harvesting of transfected cells
      • Monitoring of protein expression by YFP fluorescence
      • SDS-PAGE/Western blot analysis of protein expression

     

    Optimization

    Price includes optimization of one construct. Optimization is required before proceeding to Large-Scale Expression.
    Time required: 1.5 - 2 weeks

    Optimization of protein expression:

    • Infection of 50 mL Tni cells at one or two temperature (27 and/or 21 degrees)
    • Monitoring of protein expression time course by YFP fluorescence
    • SDS-PAGE/WESTERN analysis of expression time course

    Large-scale expression

    Once a well-expressing construct has been identified and expression conditions have been optimized, the culture volume can be scaled up. The supernatant or cell pellet is then collected from the facility.
    Time required: 1-1.5 weeks

    1. Infection of cells:

      • Infection of cells using the optimized parameters determined in the Optimization step

    2. Monitoring of protein expression:

      • Monitoring of protein expression by YFP fluorescence
      • Harvesting of cells when maximum expression level has been reached

    Size separation with Simple Western

    The Simple Western “Peggy” instrument at the CSF-ProTech can perform size separation and immunodetection of protein samples (equivalent to SDS-PAGE/Western).  The size separation range of Simple Western is from 15-150 kDa.  Larger or smaller proteins can be detected, but the molecular weight estimate will not be accurate.  Samples are run under reducing conditions. The instrument can run between 12 and 96 samples per run, in multiples of 12.  For sample analysis you must submit your samples and primary antibody.  All other consumables are included. For pricing please contact Peggy Stolt-Bergner.

    General Information

    • Simple Western runs are set up in cycles.  One cycle represents one experiment using one set of 12 samples and a primary antibody.
    • Each cycle must be run with 12 samples, consisting of 11 samples plus molecular weight ladder.
    • If you have less than 12 samples, it may be possible to combine them with other submitted samples, to reach a total of 12 per cycle.
    • One set of 12 samples can be used for multiple cycles with different antibodies.  One primary antibody can also be used in multiple cycles with different samples.
    • Several different primary antibodies may be used within one cycle, however, they should be compatible with the same secondary antibody.
    • Currently anti-mouse and anti-rabbit secondary antibodies are available.  If you need a different secondary antibody please contact the ProTech.
    • The ProTech has a small panel of primary antibodies towards common affinity and epitope tags available in house that can be requested.  To see a complete list click here.

    Before running size separation experiments on Simple Western, a few preliminary tests must be run in order to:

    • Test your antibody for compatibility with Simple Western
    • Optimize your antibody concentration
    • Optimize your sample/lysate concentration

     

    Please download the "SW_Sample_Preparation_Instructions_Size” and read the section on Antibody testing for more detailed information.

    Please also check the compatibility of the reagents in your lysis buffer in the Buffer Compatibility Chart.

     

    To submit samples for Simple Western size separation:

    1. Download and read the SW_Sample_Preparation_Instructions_Size.

    2. Download and fill out the SW_Sample_Submission_Form.

    3. Email an electronic version of your form to protech@@csf.ac.at.  You will receive an email confirmation of your order.

    4. After order confirmation, bring your samples and print-out of the form to the CSF-ProTech (1st floor, VBC2, room 1OG-10)

    5. Samples should be delivered frozen and will be kept frozen at -20 degrees C until run. (If your samples are sensitive to freezing please contact the ProTech to make other arrangements.)

    Information Request

    1. To get more information please complete all fields marked with an *.
    Electropherogram of serial dilutions of cell lysate containing a strep-tagged protein, detected with an anti-strep antibody. "Sample 1 UN" in cyan is the no primary control.

    Simple Western data is produced as an electropherogram, with one peak being equivalent to the band that one would see in a traditional Western.  The peak area can be used to perform relative quantitation of different samples.  For absolute quantitation, a standard curve of 4 different concentrations can be run and compared to experimental samples.

    On the pictures in the gallery on the right you can see a sample electropherogram for samples run on Simple Western size separation and detected with an anti-strep antibody. The electropherogram can also be converted into a "lane view" image, where the peaks in the electropherogram are presented as bands so that the representation is similar to that of a traditional Western blot.