The Elimination of Keratin Artifacts in Immunoblots Probed with Polyclonal Antibodies

Bherer, Louis; Coutu, Louise; Lefièvre, Linda; Begin, S.; Dupont, Hervé; Sullivan, Robert

doi:10.1006/abio.1994.1128

Cited by 21 publications

(3 citation statements)

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“…The streaks and faint bands are artifacts since they were also observed following electrophoresis of the sample buer alone. The appearance of such silver-staining artifacts, especially of silver-stainable contaminant bands in the molecular-weight range 54±68 kDa, has been reported repeatedly (Hashimoto et al 1983;Ochs 1983;Marshall and Williams 1984;BeÂ rubeÂ et al 1994).…”

Section: Sds-pagementioning

confidence: 92%

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Oeljeklaus

Fischer

Gerhardt

2002

Planta

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Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).

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Section: Sds-pagementioning

confidence: 92%

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Oeljeklaus

Fischer

Gerhardt

2002

Planta

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“…These Western blots were saturated for 2 h in PBS containing 10% (w:v) defatted milk and 2% normal goat serum and then incubated overnight with the anti-P34H antiserum diluted 1:1000 in PBS supplemented with 1% (v:v) goat serum. The anti-P34H polyclonal antiserum was previously adsorbed on human keratin as described by B6rub6 et al [28]. After three washes with 0.1% (v:v) Tween 20 in PBS, the nitrocellulose membranes were incubated for 1 h with a peroxidase-conjugated anti-rabbit IgG (1:3000 in PBS) and washed three times with PBS-Tween.…”

Section: P34h Sperm Protein Extractionmentioning

confidence: 99%

Surface Localization of P34H, an Epididymal Protein, during Maturation, Capacitation, and Acrosome Reaction of Human Spermatozoa1

Boué

Blais

Sullivan

1996

Biology of Reproduction

104

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During epididymal transit, spermatozoa acquire new surface antigens that are involved in the acquisition of their fertilizing ability. We have previously described a 34-kDa (P34H) human epididymal sperm protein that shows antigenic and functional homologies with the hamster P26h. P34H is localized on the acrosomal cap of human spermatozoa and has been proposed to be involved in the interaction with the zona pellucida. The aim of this study was to document the expression of P34H on the sperm surface during transit along the male and female genital tracts. Immunohistochemical techniques were performed on human testes and epididymides by means of an antiserum specific for P34H. No labelling was detected on those spermatozoa found within the seminiferous tubules or in the vasa efferentia. P34H first appeared in the caput epididymidis and was restricted to the acrosomal cap. Signal intensity then increased considerably from the proximal corpus to the cauda region of the epididymis. After ejaculation, the same pattern of P34H distribution was observed, but the intensity was much lower than that characterizing the cauda epididymal spermatozoa. Strong labeling was restored after incubation in B2 medium and was maximal after 5 h of capacitation. After acrosomal exocytosis induced by a Ca2+ ionophore, the percentage of P34H-labeled spermatozoa decreased proportionally to the number of acrosome-reacted spermatozoa as determined by Pisum sativum-fluorescein isothiocyanate (FITC) labeling. P34H appeared to be strongly anchored to the sperm plasma membrane during epididymal transit as indicated by the requirement for detergent to extract this surface antigen from ejaculated spermatozoa. This confirms the importance of P34H binding to the sperm plasma membrane during epididymal maturation. We have previously proposed that P34H is involved in sperm-zone pellucida interaction. The appearance and accumulation of P34H on the sperm plasma membrane during epididymal maturation, followed by its inaccessibility associated with ejaculation, its unmasking during capacitation, and finally its elimination after the acrosome reaction, are in agreement with te proposed function of this sperm antigen.

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“…This band is probably due to intermolecular disul®de cross-linking mediated by bmercaptoethanol (Suresh-Kumar et al, 1993), in the presence of cytokeratins in the products used for SDS-PAGE. Incubation of anti-P18 and anti-P36 sera with human cytokeratins (Be Ârube Â et al, 1994) abolished this nonspeci®c reactivity (Fig. 1c).…”

Section: Isolation Electrophoresis Immunoblotmentioning

confidence: 86%

Isolation and characterisation of two sperm membrane proteins recognised by sperm‐associated antibodies in infertile men

Auer¹,

Senechal²,

Desvaux³

et al. 2000

Mol. Reprod. Dev.

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Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.

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The Elimination of Keratin Artifacts in Immunoblots Probed with Polyclonal Antibodies

Cited by 21 publications

References 0 publications

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Surface Localization of P34H, an Epididymal Protein, during Maturation, Capacitation, and Acrosome Reaction of Human Spermatozoa1

Isolation and characterisation of two sperm membrane proteins recognised by sperm‐associated antibodies in infertile men

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