Fast horizontal electrophoresis. II. Development of fast automated staining procedures using PhastSystem™

Olsson, Ingmar; Wheeler, R. J.; Johansson, Catherine; Ekström, Björn; Stafström, Nils; Bikhabhai, Rama; Jacobson, Gunilla

doi:10.1002/elps.1150090105

Cited by 23 publications

(3 citation statements)

References 14 publications

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“…To reduce separation times while maintaining separation performance, thin‐layer IEF devices perform the separation in a thin (50–100 μm tall) polyacrylamide layer under 100–500 V/cm applied electric field strength . To confer mechanical strength, the thin polyacrylamide layers are polymerized on polyester films or glass plates.…”

Section: Introductionmentioning

confidence: 99%

“…Favorable heat dissipation is achieved owing to the high surface area to volume ratio of the thin gel layers, as is also the case in more modern microfluidic systems. To improve reproducibility and thermal management, the PhastSystem™ (Pharmacia‐LKB) features automated sample application, active cooling, voltage control, and automated staining . The PhastSystem™ completes up to 12 concurrent IEF separations per gel within a ∼15‐min assay duration.…”

Section: Introductionmentioning

confidence: 99%

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Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

2018

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Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post-separation access to the sample for downstream analysis. The two-layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post-separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self-indexed photomasks for alignment-free fabrication of multi-domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

2018

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“…To develop an efficient approach to the characterization of functional cell surface molecules, we have adapted the recently developed PhastTransfer (Pharmacia) system [4-61. This is a small format (33 X 38 mm) electrophoretic separation unit that separates 1-2 pL of sample [7]. The ultrathin gel facilitates rapid and efficient protein transfer to an immobilizing matrix [8].The efficiency of the PhastTransfer is particularly useful in the characterization of rare in vivoderived cells.…”

Section: Introductionmentioning

confidence: 99%

Semi‐dry PhastTransfer detection of biotinylated cell surface molecules

Abdi¹,

Li²,

Mentzer³

1993

Electrophoresis

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The PhastTransfer system is a semi-dry electrophoretic unit designed to optimize the transfer of small amounts of protein. Because of its efficiency, we adapted the PhastTransfer system for the detection of labeled membrane molecules. Biotin was used as the membrane molecule label because it permitted the long-term storage of labeled lysates as well as the flexibility of derivatizing several different functional groups. After immunoaffinity separation using magnetic microspheres, the protein was electrophoretically separated with the PhastSystem and transferred with the PhastTransfer unit. Using an avidin-linked enzyme amplification system, less than 10 ng of loaded protein could be detected on the transfer membrane. Based on these findings, the PhastTransfer system is a fast, reproducible, and convenient method for the transfer of small quantities of labeled cell surface protein.

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Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients

et al. 1988

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An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.

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Fast horizontal electrophoresis. II. Development of fast automated staining procedures using PhastSystem™

Cited by 23 publications

References 14 publications

Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

Arrayed isoelectric focusing using photopatterned multi‐domain hydrogels

Semi‐dry PhastTransfer detection of biotinylated cell surface molecules

Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients

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