The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes

Batteiger, Byron E.; Newhall, Wilbert J.; Jones, Robert B.

doi:10.1016/0022-1759(82)90089-8

Cited by 547 publications

(175 citation statements)

References 14 publications

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“…Samples containing 150 ,tg of cellular proteins were resolved by SDS/PAGE on 12.5 00-polyacrylamide gels and electrophoretically transferred to nitrocellulose (Towbin et al, 1979) at 0.2 A constant current overnight. Nonspecific antibody-binding sites were blocked by overnight incubation at 4°C in PBS containing 0.05 % Tween 20 (Batteiger et al, 1982 densitometer. Blotted proteins were observed after autoradiography by using Indian Ink staining (Glenney, 1986 medium was removed and the cell monolayer washed six times over 30 min at 37°C in serum-free binding medium (Dulbecco's modified Eagle's medium containing 10 mMBes and 1 mg of bovine serum albumin/ml) to remove cell-bound phorbol ester (Dunphy et al, 1980).…”

Section: Preparation Of Cell Lysates and Western Blottingmentioning

confidence: 99%

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines

Adams

Gullick

1989

Biochemical Journal

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Down-regulation of protein kinase C induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined in Swiss 3T3, V79, MDBK and C6 cells by Western blotting. Variations in the rate of down-regulation caused by treatment with 100 nM-TPA were observed; TPA treatment for 5 h caused maximal down-regulation in V79 cells, whereas TPA treatment for 10 h or 30 h was needed for maximal down-regulation of protein kinase C in MDBK or Swiss 3T3 cells respectively. The decrease in amount of immunologically detectable protein kinase C was 30% in MDBK cells and 100% in V79 and Swiss 3T3 cells. MDBK and C6 cells could be completely depleted of protein kinase C by treatment with 250 nM-TPA. In C6 cells, after treatment with 500 nM-TPA, an 80% loss of protein kinase C was seen over 10 h. Measurement of the numbers of phorbol-ester-binding sites remaining in each cell line when protein kinase C was maximally down-regulated indicated that in MDBK and Swiss 3T3 cells loss of phorbol-ester-binding sites paralleled loss of protein kinase C, whereas in V79 and C6 cells no such correlation was observed.

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Section: Preparation Of Cell Lysates and Western Blottingmentioning

confidence: 99%

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines

Adams

Gullick

1989

Biochemical Journal

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“…To determine the molecular masses of the immunoreactive polypeptides on the NC membranes, the shrinkage of the polyacrylamide gels and of the membranes during electrotransfer had to be accounted for (4). Therefore, the frozen NC membranes were allowed to thaw for 15 min at room temperature and the immobilized proteins on the membranes were visualized by staining with Ponceau S (44).…”

Section: Methodsmentioning

confidence: 99%

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

1991

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The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkenr Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labeling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.During methanogenesis from methanol and H2 by restingcell suspensions of methanol-grown Methanosarcina barkeri, ATP synthesis is stringently coupled to an electrochemical proton gradient and a functional DCCD (N,N'-dicyclohexylcarbodiimide)-sensitive ATP synthase, indicating a chemiosmotic mechanism for energy conservation (8, 10, 11). The central intermediate of methane formation from methanol is 2-(methylthio)ethanesulfonic acid (CH3-S-coenzyme M [CoM]) (19,39,43) which is reductively demrethylated with H2 to yield CH4 and 2-mercaptoethanesulfonic acid (CoM-SH) by the multicomponent methyl-coenzyme M methylreductase system (36, 37). Since the free energy change of this reaction (AG0' = -85 kJ/reaction) is large enough to drive ATP synthesis (9, 19), it has been hypothesized that one or several components of the methylreductase system are membrane associated and involved in proton translocation across the cytoplasmic membrane.It has been demonstrated recently (16,22) that the hydrogen-dependent reductive demethylation of CH3-S-CoM to CH4, a reaction common to all methanogenic bacteria irrespective of their growth substrate (19, 37), proceeds in two steps:CoM-S-S-HTP + H2 -_ CoM-SH + HTP-SH (reaction 2)In the first step (reaction 1) the methylreductase catalyzes the reduction of CH3-S-CoM to CH4 with L-7-mercaptoheptanoylthreonine phosphate (HTP-SH) as the electron donor (29). The heterodisulfide CoM-S-S-HTP formed is then reduced in the second step to CoM-SH and HTP-SH by a hydrogenase-linked heterodisulfide reductase system (reaction 2), and it has been proposed that the reduction of the mixed disulfide rather than methane formation is linked to proton translocation and thus coupled to energy conservation (16, 21).Therefore, we decided to determine the ultrastructural location of the coenzyme F420-reducing hydrogenase (F420-hydrogenase) in M. barkeri by immunoelectron microscopy, using a specific po...

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“…Proteins were separated on minigels by SDS-PAGE in 10% polyacrylamide at 20 mA per gel and transferred onto nitrocellulose membranes (Schleicher & Schuell) for 60 minutes at 0.8 mA/cm 2 by using a Semi-Dry Electroblotter (Gelman Sciences, Ancos). Nitrocellulose membranes were blocked with PBS containing 0.2% Tween 20 (Sigma) (Batteiger et al, 1982). Serum and pIgG dilutions from 10 patients and 10 controls were incubated with each membrane after addition of one sample per slot in a Cassette Miniblot System (Immunetics) for 4 hours at room temperature.…”

Section: Quantitative Immunoblotmentioning

confidence: 99%

Autoreactive Antibody Repertoire Is Perturbed in Atherosclerotic Patients

Caligiuri

Stahl

Kaveri

et al. 2003

Laboratory Investigation

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SUMMARY:In patients with clinical symptoms of coronary atherosclerosis, T cells are activated and directed to autologous proteins contained in the active plaques, suggesting that autoimmune responses may play a role in atherosclerosis progression. Organ-specific autoimmune diseases are sometimes accompanied by broad alterations of serum autoreactive antibody repertoires. We thus investigated antibody repertoires at a global level, using a technique of immunoblotting that allows for the quantitative screening of antibody reactivities in complex antibody mixtures toward a large panel of antigens derived from homologous tissue extracts, followed by multiparametric statistical analysis of the data. We analyzed the autoreactive IgG repertoire in 20 patients with documented coronary atherosclerosis and in 20 matched healthy controls. Total proteins from atherosclerotic carotid specimens and normal arterial tissues (target organs) and from kidney, liver, and stomach (non-target control organs) were used as panels of antigens. Patients had a significantly perturbed antibody repertoire and an enhanced autoreactivity of IgG to target and non-target organs, as compared with controls. Reactivity of purified IgG to plaque and normal artery proteins was greater in patients, but reactivity of IgG in the whole serum toward normal arterial tissue was lower than in controls; this suggests that, in patients, autoreactivity toward normal arteries is regulated by serum factors. Our data indicate that atherosclerotic patients develop a perturbed humoral immune response directed toward arterial proteins, which impacts on the overall autoreactive repertoire. These findings further substantiate that autoimmune processes take place in atherosclerosis and most likely influence disease progression. (Lab Invest 2003, 83:939 -947).

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The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes

Cited by 547 publications

References 14 publications

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

Autoreactive Antibody Repertoire Is Perturbed in Atherosclerotic Patients

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