Protein Crystallization

Protein crystallization is a key assay for structural studies of proteins. The protocols for crystallization of protein are challenging due to the stringent requirement for pure samples and control of environmental conditions during the crystallization process. Vapor diffusion using hanging drop is a preferred method for obtaining quality crystals with…

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ARRAYCAM® MULTIPLEX MICROARRAY IMAGING SYSTEM

Affordable bench-top microarray imager and reagent platform for protein and nucleic acid detection of microarrays printed onto ONCYTE® nitrocellulose coated chips.

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Protein Microarray Substrates

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Grace Bio-Labs microarray surface chemistry is based on the well-known protein-binding properties of nitrocellulose. A range of different formulations have…

APPLICATIONS:

Antigen-Capture AssayAntibody Capture AssayRPPA- Reverse Phase Protein MicroarrayLaser micro-dissection RRPAEpitope-mappingBiomarker Discovery and ValidationImmunogen Discovery

DNA Microarray Substrates

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Epoxy Microarray Slides provide a uniform substrate for a variety of DNA/RNA-based diagnostic applications.

APPLICATIONS:

DNA/Oligonucleotide Microarray ;  microRNA Microarray ;  Single Nucleotide Polymorphism (SNP) Analysis ;  Gene Expression Profiling; 

Microarray Reagents

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Grace Bio-Labs microarray regents have been specifically formulated to achieve the full potential of porous nitrocellulose, accelerating experimental design and…

APPLICATIONS:

Antigen-Capture Assay Antibody Capture Assay RPPA- Reverse Phase Protein Microarray Laser micro-dissection RRPA Epitope-mapping Biomarker Discovery and Validation Immunogen Discovery

APPLICATIONS:

Antibody capture assay ;  Epitope-mapping ;  Biomarker Discovery and Validation ;  Immunogen Discovery ;  Quantitative multiplex immunoassays ;  Peptide Microarrays ;  Autoantibody profiling ;  Multiplex serological assays; 

ProPlate®

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ProPlates® were specifically designed to enable automated robotic liquid handling. Two main configurations are available: The ProPlate® Microtiter Plate is comprised…

APPLICATIONS:

ProteomicsProtein MicroarraysProtein expression analysisAntibody profiling cDNA and oligonucleotide arrays

ARRAYCAM® MULTIPLEX MICROARRAY IMAGING SYSTEM

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Affordable bench-top microarray imager and reagent platform for protein and nucleic acid detection of microarrays printed onto ONCYTE® nitrocellulose coated chips.

APPLICATIONS:

Whole Proteome Screening;  Vaccine Development;  RPPA: Reverse Phase Protein Array;  Biomarker Discovery and Validation;  Epitope Mapping; 

NanoParticle Fluorescent Calibration Slide

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Photostable nanoparticles arrayed on glass slides for calibration of fluorescence imaging systems and quantitative analysis.

APPLICATIONS:

Calibration of Microarray Scanners ;  Quantitative Microarray Analyses Microscope Focal Plane Adjustment;  Microscope Focal Plane Adjustment; 

CoverWell Perfusion Chambers

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CoverWell ™ perfusion press-to-seal covers form water-tight, multiwell cell incubation or cytochemistry chambers when pressed to coverslips or microscope slides.…

APPLICATIONS:

Live-cell imagingMicroscopyImagingSingle molecule spectroscopy

SecureSeal™ Hybridization Chambers

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SecureSeal™ Hybridization Chambers are thin, silicone-gasketed chambers providing optimal surface-to-volume fluid dynamics for hybridization assays on large or multiple specimens…

APPLICATIONS:

In situ hybridizationProtein and DNA MicroarraysImmunocytochemistryRapid microfluidic prototypingFluorescence Resonance Energy Transfer (FRET)

CoverWell™ Incubation Chambers

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CoverWell™ incubation chambers are reusable, easy to apply chambers that attach without the use of adhesive.  CoverWells™ enclose a large…

APPLICATIONS:

Reverse Transfection Microarray;  DNA Microarray;  In-situ hybridization;  Immunohistochemistry; 

Silicone Isolators

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Silicone Isolators allow researchers to isolate specimens using removable hydrophobic barriers. They may be used to isolate cells grown in…

SecureSeal™ Hybridization Chambers

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SecureSeal™ Hybridization Chambers are thin, silicone-gasketed chambers providing optimal surface-to-volume fluid dynamics for hybridization assays on large or multiple specimens…

HybriWell™ Sealing System

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HybriWell™ Sealing System bonds securely to a microscope slide surface in seconds to confine small reagent volumes with samples and…

Hybridization and Incubation

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Hybridization and incubation Seals ad Chambers from Grace Bio-Labs are ideally suited for in situ-hybridization assays. The adhesive seal of…

APPLICATIONS:

In-situ hybridization MicroarraysFluorescence In situ Hybridization (FISH)FRET (Fluorescence Resonance Energy Transfer)

FastWells™ Reagent Barriers

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FastWells™ are sticky, flexible silicone gaskets that form hydrophobic reagent barriers around specimens without messy adhesives or special slides. Gaskets may…

FlexWell™ Incubation Chambers

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FlexWell™ incubation chamber silicone gaskets form wells on slides using clean release adhesive to isolate up to 16 specimens per…

APPLICATIONS:

Protein MicroarrayHybridizationIncubation

HybriSlip™ Hybridization Covers

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HybriSlips™ are rigid, light-weight, thin plastic coverslips that minimize friction and facilitate uniform reagent distribution during incubation steps which require…

ProPlates®

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ProPlates® were specifically designed to enable automated robotic liquid handling. Two main configurations are available: The ProPlate® Microtiter Plate is comprised…

APPLICATIONS:

ProteomicsProtein MicroarraysProtein expression analysis;  Antibody profiling ;  cDNA and oligonucleotide arrays; 

Silicone Isolators™ Sheet Material

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Silicone isolator™ sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. Where additional sealing is…

APPLICATIONS:

Protein and DNA arrays ;  Immunohistochemistry;  Fluorescence In situ Hybridization (FISH) ;  Biopolymers and hydrogel formulation ;  Cryogenic-transmission electron microscopy (Cryo-TEM) ;  Microwave crystallization ;  Ultra-small-angle X-ray scattering (USAXS) ;  Tissue ingeneering;  Live cell lithography” (LCL); 

Imaging Spacers

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Imaging spacers are ultra-thin adhesive spacers which peel-and-stick to coverglass or microscope slides to confine specimens without compression. Layer multiple…

APPLICATIONS:

Imaging;  Microscopy;  High-temperature single-molecule kinetic analysis;  Anti‐Stokes Raman scattering microscopy; 

CoverWell™ Imaging Chambers

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CoverWell ™ imaging chambers are designed to stabilize and support thick and free-floating specimens for confocal microscopy and imaging applications.…

APPLICATIONS:

Confocal microscopy Imaging Tissue and Cell staining ;  High Resolution Microscopy ;  Live-cell imaging ; 

CoverWell™ Perfusion Chambers

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CoverWell ™ perfusion press-to-seal covers form water-tight, multiwell cell incubation or cytochemistry chambers when pressed to coverslips or microscope slides.…

APPLICATIONS:

Single molecule spectroscopy Live-cell imaging Microscoscopy

FastWells™ Reagent Barriers

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FastWells™ are sticky, flexible silicone gaskets that form hydrophobic reagent barriers around specimens without messy adhesives or special slides. Gaskets may…

APPLICATIONS:

Microscopy Fluorescence In situ Hybridization (FISH) Single-molecule fluorescence analysis ;  Immunohistochemistry ; 

MultiSlip™ Coverglass Inserts

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MutliSlip™ inserts with 8 (18mm x 18mm) or 15 (12mm x 12mm) No. 1.5 German glass coverglass per insert are…

APPLICATIONS:

High resolution microscopy Fluorescent imaging Immunohistochemistry ;  Cell Culture; 

SecureSeal™ Adhesive Sheets

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These adhesive sheets are made using the same SecureSeal™ adhesive as is used to make HybriWell™ and SecureSeal™ Incubation Chambers.  Thin,…

APPLICATIONS:

Imaging ;  Tissue and Cell staining ;  High Resolution Microscopy; 

SecureSlip™ Silicone Supported Coverglass

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SecureSlip™ Silicone Supported Coverglass is affixed to a thin microscopically transparent silicone base which secures it to culture vessels by…

Imaging and Microscopy

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Imaging seals and chambers from Grace Bio-Labs offer a selection of tools for cell/tissue staining for high quality results in…

APPLICATIONS:

Tissue and Cell stainingHigh Resolution MicroscopyLive-cell imaging

CultureWell™ MultiWell Chambered Coverslips

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CultureWell™ chambered coverglass products consist of removable and reusable, non-cytotoxic silicone gaskets secured to number 1.5 German coverglass. Chambered coverglass…

APPLICATIONS:

Cell Culture Fluorescence applications In-situ hybridization Immunostaining

CS16-CultureWell™ Removable Chambered Coverglass

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CS16 CultureWell™ removable chambered coverglass is a 16-well chambered coverglass cell culture vessel, with 2 x 8 format with standard…

APPLICATIONS:

Cell CultureFluorescence applicationsIn-situ hybridizationImmunostaining

CultureWell™ Coverglass Inserts

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Each CultureWell™ coverglass insert is comprised of four chambered coverglass, assembled in a disposable frame placed in a standard 86mm…

APPLICATIONS:

High resolution microscopy Fluorescent imaging Immunohistochemistry

CultureWell™ Reusable Gaskets

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Gaskets are ideal for forming wells on glass microscope slides or in polystyrene dishes. Gaskets are non-sterile and may be…

APPLICATIONS:

Cell CultureHigh resolution microscopyFluorescent imaging Immunohistochemistry

CultureWell™ Silicone Sheet Material

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CultureWell™ clear silicone sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. They may be…

APPLICATIONS:

Cell CultureHigh resolution microscopy Fluorescent imagingImmunohistochemistry

MultiSlip™ Coverglass Inserts

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MutliSlip™ inserts with 8 (18mm x 18mm) or 15 (12mm x 12mm) No. 1.5 German glass coverglass per insert are…

APPLICATIONS:

Cell CultureFluorescent imaging Immunohistochemistry

SecureSlip™ Silicone Supported Coverglass

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SecureSlip™ Silicone Supported Coverglass is affixed to a thin microscopically transparent silicone base which secures it to culture vessels by…

APPLICATIONS:

Cell CultureImmunofluorescence assayMicroscopy

CultureWell™ ChamberSLIP 16, Non-Removable Chambered Coverglass

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CultureWell™ NON Removable Chambered Coverglass, 16 Well, No. 1.5 German borosilicate Coverglass. Product consists of cell culture vessels, with a…

APPLICATIONS:

Cell Culture Fluorescence applicationsSmall volume incubation Immunostaining

Silicone Wound Splints

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Wound splints are constructed of silicone and include suture sites for increased precision in affixing on or within an animal…

Silicone Isolator Sheet Material

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Silicone isolator™ sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. Where additional sealing is…

APPLICATIONS:

Protein and DNA arrays ;  Immunohistochemistry ;  Fluorescence In-situ Hybridization (FISH) ;  Biopolymers and hydrogel formulation;  Cryogenic-transmission electron microscopy (Cryo-TEM) ;  X-ray scattering ;  Microwave crystallization ;  Ultra-small-angle X-ray scattering (USAXS) ;  Tissue engineering Live cell lithography (LCL); 

CultureWell Silicone Sheet Material

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CultureWell™ clear silicone sheet material allows researchers to create their own removable hydrophobic barriers to isolate specimens. They may be…

APPLICATIONS:

Lorem Ipsum ;  Lorem Ipsum;  Lorem Ipsum; 

BIOLOGICAL COATING PROCEDURES

Biological coating procedures for CultureWell TM, MultiSlip TM and SecureSlip TM glass coverslips.

Collagen may be used to coat glass coverslips for the growth of epithelial, endothelial and muscle cells, neurons, PC12 and CHO cell lines. Type I collagen is most often used for coating substrates for cell culture because it is easily obtainable from rat tails. For short term cultures, collagen can be simply applied to glass coverslips and allowed to dry.

  1. Dilute collagen solution 1:10 – 1:50 with 30% ethanol and spread over surface of sterile glass coverslip.
  2. Air dry in a tissue culture hood.
  3. Cells can be seeded directly on the collagen surface.
  4. Collagen coating prepared in this way tends to detach from the glass in long-term cultures.

Collagen IV is the major constituent of basement membrane and is, therefore, a more physiological coating for the culture of many cell types. For long-term cultures, collagen I and IV can be applied to glass coverslips by first coating the glass with polylysine or polyornithine. This provides a more stable collagen coating.

  1. Prepare polylysine or polyornithine (MW of 30,000 – 70,000) at 0.1-1 mg/ml in 0.15 M borate buffer (pH 8.3). Filter sterilize.
  2. Add enough solution to pool over surface of sterile glass coverslip.
  3. Incubate 2-24 hours at room temperature.
  4. Aspirate solution and wash coverslips 3 times with water.
    Pool collagen solution, 100 ug/ml in water over surface of coverslip.
  5. Incubate 4 – 16 hours.
  6. Rinse once with media and seed with cells.

Alternatively, for long-term cultures, double layered collagen coatings can provide a stable coating.

  1. Spread a couple of drops of sterile collagen I solution on the sterile glass coverslip.
  2. Immediately neutralize for 2 minutes with ammonium hydroxide vapors by placing the dish of coverslips in a covered dish containing filter paper wet with concentrated ammonium hydroxide. This will cause the collagen to gel.
  3. Wash coverslips twice with sterile water.
  4. Gently spread a couple of drops of collagen over the surface of the gelled collagen and air dry.
  5. Use within a few hours for cell culture.

Gelatin can also be used for the culture of some cell types including glial cells.

  1. Dissolve 100 mg gelatin in 100 ml water (triple glass distilled or RO).
  2. Autoclave to sterilize.
  3. While hot, thoroughly mix gelatin solution.
  4. Add enough solution to pool over surface of sterile glass coverslip.
  5. Chill for 2-24 hours at 4oC.
  6. Remove gelatin by aspiration and add sterile water.
  7. Dishes can be stored for up to one week at 4oC.
  8. Remove water immediately before use for cell culture.

Nearly all types of cells adhere to these polymers of basic amino acids. They are particularly useful for the culture of CNS neurons. The L- or D-isomers can be used for cell attachment, however, the D-isomer may be preferred because it is not subject to breakdown by proteases released by cells.

  1. Prepare polylysine or polyornithine (MW of 30,000 – 70,000) at 0.1-1 mg/ml in 0.15 M borate buffer (pH 8.3). Filter sterilize.
  2. Add enough solution to pool over surface of sterile glass coverslip.
  3. Incubate 2-24 hours at room temperature.
  4. Aspirate solution and wash coverslips 3 times with media or PBS.
  5. Immediately add cell suspension or growth media.

Fibronectin is an extracellular matrix constituent use for the culture of endothelial cells, fibroblasts, neurons and CHO cells.

  1. Stock solution can be prepared by dissolving 1 mg/ml fibronectin in PBS. Filter sterilize and freeze in aliquots.
  2. Diluted stock solution to 50-100 ug/ml in basal medium or PBS.
  3. Add enough solution to pool over surface of sterile glass coverslip.
  4. Incubate for 30-45 min at room temperature.
  5. Aspirate to remove fibronectin and rinse coverslips with media or PBS.
  6. Immediately add cell suspension or growth media. Do not allow coating to dry.

Laminin is an extracellular matrix constituent used for the culture of neurons, epithelial cells, leukocytes, myoblasts and CHO cells.

  1. Stock solution can be prepared by dissolving 1 mg/ml laminin in PBS. Filter sterilize and freeze in aliquots.
  2. Diluted stock solution to 10-100 ug/ml in basal medium or PBS.
  3. Add enough solution to pool over surface of sterile glass coverslip.
  4. Incubate several hours at room temperature.
  5. Aspirate to remove laminin and rinse coverslips with media or PBS.
  6. Immediately add cell suspension or growth media. Do not allow coating to dry.
  7. Coating the glass coverslip first with polylysine or polyornithine and then laminin may increase the concentration of laminin applied using this method.

RECOMMENDED FOR LABORATORY USE ONLY

 

 

FAST GREEN STAINING OF ONCYTE® POROUS NITROCELLULOSE FILM SLIDES WITH NEAR IR DETECTION

Grace Bio-Labs Laboratory Method

FAST Green Staining of ONCYTE® Porous Nitrocellulose Film Slides with near IR Detection See Original Article

1.1    General staining of proteins deposited on porous nitrocellulose films for sample protein quantification.

2.1    Equipment

  2.1.1        Orbital shaker

  2.1.2        ArrayCAM™ Microarray Imager (Grace Bio-Labs)

2.2    Materials and Reagents

2.2.1        Glass staining dishes with lids (Wheaton Cat# 900203).

2.2.2        Glass slide racks with handles (Wheaton Cat# 900204, 900205).

2.2.3        ProPlate™ modules (Grace Bio-Labs)

2.2.4        Fast Green FCF (Sigma-Aldrich Cat#F7258)

2.2.5        Methanol

2.2.6        Glacial Acetic Acid

2.2.7        Distilled Water

2.3    Solutions

2.3.1        De-Staining Solution (1000mL):

2.3.1.1  Methanol (300mL, 30%)

2.3.1.2  Glacial Acetic Acid (70mL, 7%)

2.3.1.3  Water (630mL, 63%)

2.3.2        Fast Green Stock Solution (400x, 10mL)

2.3.2.1  Fast Green (0.1g)

2.3.2.2  De-Staining Solution (9.9mL)

2.3.3        Fast Green Staining Solution (1x, 1000mL)

2.3.3.1  Fast Green Stock Solution (2.5mL)

2.3.3.2  De-Staining Solution (997.5mL)

2.3.4        1% NaOH

3.1    Transfer slides to a staining jar containing fresh distilled water. 

3.1.1        Wash for 5 minutes with agitation (105 rpm on orbital shaker).

3.2    Transfer slides to a staining jar containing 1% NaOH.

3.2.1        Incubate for 15 minutes with agitation (105 rpm on orbital shaker).

3.3    Transfer slides to a staining jar containing fresh distilled water.

3.3.1        Rinse briefly by submerging slides repeatedly 10-20 times over 1 minute.

3.4    Transfer slides to a staining jar containing fresh distilled water.

3.4.1        Wash for 10 minutes with agitation (105 rpm on orbital shaker).

3.5    Transfer slides to a staining jar containing De-Staining Solution.

3.5.1        Wash for 15 minutes with agitation (105 rpm on orbital shaker).

3.6    Transfer slides to a staining jar containing Fast Green Staining Solution.

3.6.1        Wash for 3 minutes with agitation (105 rpm on orbital shaker).

3.7    Transfer slides to a staining jar containing fresh water.

3.7.1        Rinse briefly by submerging slides repeatedly 10-20 times over 1 minute.

3.8    Transfer slides to a staining jar containing De-Staining Solution.

3.8.1        Wash for 15 minutes with agitation (105 rpm on orbital shaker)

3.9    Transfer slides to a staining jar containing fresh water.

3.9.1        Rinse briefly by submerging slides repeatedly 10-20 times over 1 minute.

3.10  Dry the slide by centrifugation.

3.11  Scan slides using a fluorescent imager capable of measuring at 800 nm (settings and results may vary with imaging system and filters employed):

3.11.1    ArrayCAM Microarray Imager

3.11.1.1                      800 nm bandpass

3.11.1.2                      Typical Settings:

6.2.1.2.1        Exposure: 200 msec.

6.2.1.2.2        Acquisition Time: 4 sec.

6.2.1.2.3        Gain: 20%

3.12 See Appendix for typical results obtained with this method.

4.1    Loebke, et al. (2007) Infrared-based protein detection arrays for quantitative proteomics. Proteomics 7, 558-584.

4.2    Levine, et al. (2006) Quantitation of protein on gels and blots by infrared fluorescence of Coomasie blue and Fast Green. Analytical Biochemistry 350, 233- 238.

Figure 1. Fast Green Protein Quantification on ONCYTE® Film Slides with near IR detection using ArrayCam.

Figure 2. Quantitation of Cell Lysates on RPPA using Fast Green with ArrayCAM produces results comparable to Sypro Ruby Protein Stain measured on a GenePix scanner (Molecular Devices, Sunnyvale, CA).

Figure Legends:

Figure 1.  Total protein may be quantified after spotting on ONCYTE® AVID Film Slides by staining with Fast Green and measuring fluorescence using an ArrayCAM, an imaging system developed at Grace Bio-Labs.   ArrayCAM is a CCD-based imaging system using laser excitation at 405 nm. Fast Green emission is 800 nm. (NOTE: Similar results were obtained with ONCYTE® SuperNOVA Film Slides from Grace Bio-Labs.)

A.  Shown is a range of standard proteins from 8 – 500 pg after on-slide staining with Fast Green.

B.  Detection and quantitation of BSA, Lysozyme, and IgG using a Fast Green staining protocol (see attachment) with ArrayCAM are linear down to the lowest protein deposition (8 pg). 

 

Figure 2. Fluorescent signal for total protein detection with Fast Green/ArrayCAM yields linear results at cell lysate concentrations commonly used for RPPA analysis down to approximately 8 pg protein per spot.   Similar results were obtained using a standard protein stain method (SYPRO® Ruby total protein stain; 532nm/575nm detection) with the use of a focused-laser microarray scanner (Molecular Devices GenePix 4400).  Cell lysates were from Calyculin A-treated Jurkat cells spotted in 2-fold serial dilution starting with a concentration of 1 mg/ml in a Tris/SDS/Glycerol lysis buffer.

Download Fast Green Protocol