An improved method for immobilizing IgG antibodies on protein A-agarose

Sisson, Thomas H.; Castor, C. William

doi:10.1016/0022-1759(90)90071-3

Cited by 70 publications

(29 citation statements)

References 6 publications

(4 reference statements)

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“…Following centrifugation (14,000g for 2 min at 4°C) 5 lg of the capture antibody (mAb 1/B1 or P28) was added to the cold precleared supernatant (the optimal concentration of antibody was determined by titration) and incubated for 1 h at 4°C. Then, 50 ll Protein G Agarose was added to the samples and incubated on a rocking platform overnight at 4°C [29]. The beads were collected with centrifugation (14,000g for 2 min at 4°C) and washed three times with 50 mM Tris-HCl, 145 mM NaCl, 1% Triton X-100, pH 7.2.…”

Section: Monoclonal Human Trypsinogen 4 Antibodiesmentioning

confidence: 99%

Regional Distribution of Human Trypsinogen 4 in Human Brain at mRNA and Protein Level

et al. 2007

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Gene PRSS3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. Mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mRNA for trypsinogen 4 has recently been identified in brain and other human tissues. We measured the amount of trypsinogen 4 mRNA and the quantity of the protein as well in 17 selected areas of the human brain. Our data suggest that human trypsinogen 4 is widely but unevenly distributed in the human brain. By immunohistochemistry, here we show that this protease is localized in neurons and glial cells, predominantly in astrocytes. In addition to cellular immunoreactivity, human trypsinogen 4 immunopositive dots were detected in the extracellular matrix, supporting the view that human trypsinogen 4 might be released from the cells under special conditions.

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Section: Monoclonal Human Trypsinogen 4 Antibodiesmentioning

confidence: 99%

Regional Distribution of Human Trypsinogen 4 in Human Brain at mRNA and Protein Level

et al. 2007

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“…Lysates were ultracentrifuged at 100,000 g for 30 minutes at 4°C and the pH was then adjusted to 7 with Tris-HCl. Pre-immune or affinity-purified anti-Sam68 AD1 antibodies were covalently coupled to protein G-Sepharose by dimethylpimelimidate-mediated cross-linking (Sisson and Castor, 1990). Each resin was incubated for 2 hours with neuron lysates, at 4°C, washed with Tris 10 mM pH 7/1% Triton X-100 and then subjected to SDS-PAGE.…”

Section: Cell Stimulationmentioning

confidence: 99%

Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons

Fredj

Grange

Sadoul

et al. 2004

Journal of Cell Science

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The traffic and expression of mRNAs in neurons are modulated by changes in neuronal activity. The regulation of neuronal RNA-binding proteins is therefore currently receiving attention. Sam68 is a ubiquitous nuclear RNA-binding protein implicated in post-transcriptional processes such as signal-dependent splice site selection. We show that Sam68 undergoes activity-responsive translocation to the soma and dendrites of hippocampal neurons in primary culture. In unstimulated neurons transiently expressing a GFP-Sam68 fusion protein, 90% of the cells accumulated the protein exclusively in the nucleus, and 4% showed extension of GFP-Sam68 to the dendrites. This nuclear expression pattern required the integrity of the Sam68 N-terminus. When present, the dendritic GFP-Sam68 formed granules, 26% of which were colocalized with ethidium bromide-stained RNA clusters. Most of the GFP-Sam68 granules were completely stationary, but a few moved in either a retrograde or anterograde direction. Following depolarization by 25 mM KCl, 50% of neurons displayed dendritic GFP-Sam68. GFP-Sam68 invaded the dendrites after 2 hours with high KCl, and returned to the nucleus within 3 hours after termination of the KCl treatment. A control GFP fusion derived from the SC-35 splicing factor remained fully nuclear during depolarization. No significant change was observed in the phosphorylation of Sam68 after depolarization. Translocation of Sam68 to the distal dendrites was microtubule dependent. Blockade of calcium channels with nimodipine abolished the translocation. Furthermore, inhibition of CRM-1-mediated nuclear export by leptomycin B partially prevented the depolarization-induced nuclear efflux of GFP-Sam68. These results support the possible involvement of Sam68 in the activity-dependent regulation of dendritic mRNAs.

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“…Generally, this method is used when a high capacity/high activity support is needed. If a more permanent immobilization is desired, the adsorbed antibodies may be cross-linked to the support material using carbodiimide (Phillips et al, 1985) or dimethyl pimelimidate (Schneider et al, 1982;Sisson & Castor, 1990).…”

Section: Immunoglobulin Purification (Antibody Immobilization)mentioning

confidence: 99%