A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins

Oliver W. Press; Ellen S. Vitetta; Paul J. Martin

doi:10.1016/0165-2478(86)90017-9

A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins

Oliver W. Press, Ellen S. Vitetta, Paul J. Martin

Research output: Contribution to journal › Article › peer-review

14 Scopus citations

Abstract

We describe a simplified cell free reticulocyte lysate protein synthesis assay which has been shown to reliably demonstrate dose dependent inhibition of protein synthesis with cycloheximide and with four ricin A chain immunotoxin conjugates. Maximal [³H]leucine incorporation was observed using 40 μl of reticulocyte lysate per microtiter well and by incubating samples for 30 minutes before harvesting with a multiple automated sample harvester. Trichloroacetic acid precipitation and hydrogen peroxide decolorization were not required with this method. This rapid, simplified assay system will facilitate testing of agents that inhibit protein synthesis, particularly immunotoxins.

Original language	English (US)
Pages (from-to)	37-41
Number of pages	5
Journal	Immunology Letters
Volume	14
Issue number	1
DOIs	https://doi.org/10.1016/0165-2478(86)90017-9
State	Published - Nov 17 1986

Keywords

Immunotoxin
Protein synthesis
Reticulocyte Lysate
Ricin
Toxin

ASJC Scopus subject areas

Immunology and Allergy
Immunology

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10.1016/0165-2478(86)90017-9

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title = "A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins",

abstract = "We describe a simplified cell free reticulocyte lysate protein synthesis assay which has been shown to reliably demonstrate dose dependent inhibition of protein synthesis with cycloheximide and with four ricin A chain immunotoxin conjugates. Maximal [3H]leucine incorporation was observed using 40 μl of reticulocyte lysate per microtiter well and by incubating samples for 30 minutes before harvesting with a multiple automated sample harvester. Trichloroacetic acid precipitation and hydrogen peroxide decolorization were not required with this method. This rapid, simplified assay system will facilitate testing of agents that inhibit protein synthesis, particularly immunotoxins.",

keywords = "Immunotoxin, Protein synthesis, Reticulocyte Lysate, Ricin, Toxin",

author = "Press, {Oliver W.} and Vitetta, {Ellen S.} and Martin, {Paul J.}",

note = "Funding Information: * Supported by American Cancer Society Institulional Grant Award No. IN-26Z, Contract N0014-82-K0660 from the Office of Naval Research, Department of Defense, and NIH Grant CA-28149. Dr. Press is the recipient of a Clinical Oncology Ca-reer Development Award (No. 85-35) from the American Can-cer Society.",

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doi = "10.1016/0165-2478(86)90017-9",

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T1 - A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins

AU - Press, Oliver W.

AU - Vitetta, Ellen S.

AU - Martin, Paul J.

N1 - Funding Information: * Supported by American Cancer Society Institulional Grant Award No. IN-26Z, Contract N0014-82-K0660 from the Office of Naval Research, Department of Defense, and NIH Grant CA-28149. Dr. Press is the recipient of a Clinical Oncology Ca-reer Development Award (No. 85-35) from the American Can-cer Society.

PY - 1986/11/17

Y1 - 1986/11/17

N2 - We describe a simplified cell free reticulocyte lysate protein synthesis assay which has been shown to reliably demonstrate dose dependent inhibition of protein synthesis with cycloheximide and with four ricin A chain immunotoxin conjugates. Maximal [3H]leucine incorporation was observed using 40 μl of reticulocyte lysate per microtiter well and by incubating samples for 30 minutes before harvesting with a multiple automated sample harvester. Trichloroacetic acid precipitation and hydrogen peroxide decolorization were not required with this method. This rapid, simplified assay system will facilitate testing of agents that inhibit protein synthesis, particularly immunotoxins.

AB - We describe a simplified cell free reticulocyte lysate protein synthesis assay which has been shown to reliably demonstrate dose dependent inhibition of protein synthesis with cycloheximide and with four ricin A chain immunotoxin conjugates. Maximal [3H]leucine incorporation was observed using 40 μl of reticulocyte lysate per microtiter well and by incubating samples for 30 minutes before harvesting with a multiple automated sample harvester. Trichloroacetic acid precipitation and hydrogen peroxide decolorization were not required with this method. This rapid, simplified assay system will facilitate testing of agents that inhibit protein synthesis, particularly immunotoxins.

KW - Immunotoxin

KW - Protein synthesis

KW - Reticulocyte Lysate

KW - Ricin

KW - Toxin

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A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins

Abstract

Keywords

ASJC Scopus subject areas

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