Partial characterization of three hemolymph proteins of Penaeus semisulcatus de haan (crustacea, decapoda, penaeidae) and their specific antibodies

Tom, Moshe; Shenker, Ofer; Ovadia, Michael

doi:10.1016/0305-0491(93)90217-s

Cited by 6 publications

(2 citation statements)

References 27 publications

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“…After this initial MS identification, we considered the crossreactivity of the crayfish proteins by western blot analysis using anti-hemocyanin antiserum raised against hemolymph hemocyanin of the shrimp Penaeus semisulcatus (Tom et al, 1993) (Fig.2A). The antibodies cross-reacted with both hemolymph and gastrolith hemocyanins from our crayfish but not with BSA, a negative control.…”

Section: Resultsmentioning

confidence: 99%

“…The membrane was blocked with 5% skimmed milk in Tris-buffered saline (TBS) then incubated with anti-hemocyanin antisera (Tom et al, 1993) at a dilution of 1:1000 (v/v). After washing with TBS containing 0.1% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (1:15,000, v/v).…”

Section: Western Blot Analysismentioning

confidence: 99%

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Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith

Glazer

Tom

Weil

et al. 2013

Journal of Experimental Biology

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SUMMARYGastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

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Section: Resultsmentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%