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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Buffers and Reagents

High-quality, ready-to-use buffers and reagents manufactured in ISO- certified facilities.

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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…

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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.

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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…

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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…

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ProteomicsProtein MicroarraysProtein expression analysisAntibody profiling cDNA and oligonucleotide arrays

NanoParticle Fluorescent Calibration Slide

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

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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.…

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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…

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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…

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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…

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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…

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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…

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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); 

CultureWell Removable chamber slide 8 well Catalog #: 103542

Microscopy Reagents

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Grace Bio-Labs microscopy reagents are manufactured in ISO-certified facilities to ensure the highest quality and consistency.

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…

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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.…

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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.…

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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…

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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…

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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,…

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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…

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Tissue and Cell stainingHigh Resolution MicroscopyLive-cell imaging

CultureWell Removable chamber slide 8 well Catalog #: 103542

Cell Culture Reagents

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Grace Bio-Labs microscopy reagents are manufactured in ISO-certified facilities to ensure the highest quality and consistency.

CultureWell removable chamber slide

CultureWell™ Removable Chamber Slide

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CultureWell- removable chamber slide allows the cultivation and analysis of cells directly on a borosilicate microscope slide.

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Fluorescence microscopy ;  Confocal microscopy ;  Cell differentiation and transfection;  Immunohistochemistry ;  Immunofluorescence;  Immunostaining;  Tissue and cell staining ;  Fluorescence Resonance Energy Transfer (FRET) Microscopy; 

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…

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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…

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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…

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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…

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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…

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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…

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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…

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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…

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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…

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Lorem Ipsum ;  Lorem Ipsum;  Lorem Ipsum; 

From Western Blot to Protein Array with Super G Blocking Buffer

IN Featured Research, Product Applications, Super G, Uncategorized

logo gracebiolabsResults obtained from Western blots can be highly variable and dependent not only on the specific antigen/antibody pairs used, but also on other reagents in the protocol such as blocking buffer. Not surprisingly, no one “universal” blocking reagent works well with all proteins. Thus choice of blocking reagent should be assessed for optimal results and having various blockers available in your lab is an advantage. We show here results of Western Blots using Super G blocking buffer.

Super G blocking buffer, although designed for microarray applications, is an effective blocker for Western blot assays on both nitrocellulose and PVDF membranes.  Its non-protein based formula was designed for high sensitivity applications on porous membranes.  Presented  here are results obtained with Western blots after blocking with Super G for the detection of various proteins (GAPDH, PARP, b-Actin, Histone H2A, MAPK). Super G generally produced results comparable to those obtained with a common casein blocker, though in some cases one blocking reagent or another performed better for a specific protein.  For example, after blocking with casein Histone H2A barely detected, requiring an extended development time while blocking with  Super G produced superior results. In situations such as this when long developing times are required for obtaining visible signal, background levels will also be elevated significantly and may obscure results. In contrast, blots for MAPK required longer development after Super G blocking to obtain comparable results to casein.  Further optimization (decreasing blocking time) may remedy this and is on-going further trials with Super G, but speaks to the variability expected from antigen to antigen with different blockers. 

Super G Blocking Buffer is compatible with direct detection methods such as fluorescence as well as enzyme-linked detection methods using alkaline phosphatase (as shown here) and horseradish peroxidase (data not shown).  It is very efficient at blocking porous membranes and, in most cases, dilutions up to 10-fold in 1x PBS were used for adequate blocking.  Additionally, shorter blocking times are possible down to 10 min. (data not shown).  In general, blocking with Super G produced excellent results for Western blots and may facilitate detection for some proteins over other blockers. 

Figure 1. Detection by Fluorescence and Colorimetric Methods on PVDF Membranes

 

Detection by Fluorescence and Colorimetric Methods on PVDF Membranes

 

Figure 1. Typical results achieved with Super G blocking reagent compared to casein blocking on Western blots using fluorescent and colorimetric endpoint detection on PVDF membranes. For all panels, 500 ng of mouse liver lysates were separated by SDS-PAGE and transferred to Millipore Immobilon-FL (fluorescent) and Immobilon-P (colorimetric) PVDF membranes.  Membranes were blocked for 1 hour with respective blockers (Panel A: 0.1x Super G in 1x PBS; B: Thermo Casein Blocker; C: 0.5x Super G; D: 0.25x Super G; E: 0.1x Super G). Blocking was followed by detection with a monoclonal anti-GAPDH antibody (1:1000) overnight in 1x PBST followed with (A) 1:10,000 anti-goat IgG-Alexa-647 and –Alexa555 or (B-E) colorimetric endpoint detection utilizing alkaline phosphatase with BCIP/NBT substrate (VECTASTAIN ABC System, Vector Laboratories) after a 5 minute development time.  A band at 37 kDa is detectable on all blots.

 

Figure 2. Detection by Colorimetric Methods on Nitrocellulose Membranes

Detection by Colorimetric Methods on Nitrocellulose Membranes

 

Figure 2. Typical results achieved with Super G blocking reagent compared to casein blocking on Western blots using colorimetric endpoint detection on nitrocellulose membranes. For all panels, 500 ng of mouse liver lysates were separated by SDS-PAGE and transferred to Whatman Protran nitrocellulose membranes.  Membranes were blocked for 1 hour with respective blockers (0.1x Super G in 1x PBS or Thermo Casein Blocker) followed by detection with various antibodies (1:1000) overnight in 1x PBST/0.25% BSA (Panel A: monoclonal anti-p44/42 MAPK; Panel B: polyclonal anti-b-Actin; Panel C: monoclonal anti-PARP; Panel D: polyclonal anti-Histone H2A). Visualization was performed with colorimetric endpoint detection utilizing alkaline phosphatase with BCIP/NBT substrate (VECTASTAIN ABC System).  Development times varied for antibody and blocker as follows: Panel A – 60 min. (Super G) and 8 min. (casein); Panel B – 10 min. (Super G) and 3 min. (casein); Panel C – 15 min. (Super G) and 5 min. (casein); and Panel D – 15 min. (Super G) and 93 min. (casein).  Expected bands were visible for all blots: (A) 42 and 44 kDa MAPK, (B) 45 kDa b- Actin, (C) 116 kDa PARP, and (D) 12 kDa Histone H2A.