PROTEIN PROFILING PECTORAL FIN-LOSS OF ADULT AFRICAN CATFISH Clarias gariepinus AND IT’S EXPRESSION OF T-Box5 PROTEIN
Description
Various studies on aquatic commodity abnormalities have been studied since 48 years ago (Aulstad & Kittelsen, 1971) but the problem of skeletal abnormalities in fish cultivation is still a major obstacle for the aquaculture sector (Bardon et al, 2009; Estivals, et al, 2015) . Skeletal abnormalities are still a stagnant problem (Boglione et al, 2013). African Catfish Clarias gariepinus is a popular species and has been widely cultivated in Indonesia since the 1980s. The hatchery technology has been mastered and is easy to do, making this fish more popular. In some centers of fish cultivation, abnormal fish are found in various forms of abnormalities. One form of abnormality that occurs is pectoral fin abnormality. It was suspected that the loss of the C.gariepinus African pectoral fin organ from the origin of cultivation in Indonesia was caused by genetic factors. This refers to the knowledge that pectoral fin organogenesis involves genes that work together so that organs can grow in the right place and pair with symmetric bilateral forms. Searching for the activity of pectoral fin organ formation genes, it was found that the gene that determines the pectoral fin identity is the Tbx5 gene (Pi-Roiget al, 2014; Albalat et al, 2010; Hall, 2005; Minguillon et al, 2005; Begemann and Ingenham , 1999; Tamura et al, 1999). This study aims to obtain the expression of protein T-Box5 (TBX5) of C.gariepinus African catfish that have pectoral fin morphological abnormalities. If TBX5 expression is different between abnormal fish and normal fish (control), then it can be proved that the pectoral fin morphological abnormality is a contribution from internal factors, in this case genetic factors. This research needs to be done so that the assessment of abnormalities in fish cultivation will progress, especially in the genetic field, so that a solution can be formulated to overcome the existing problems.
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Extraction and Isolation of protein Isolation of organs on an ice block was carried out using sectional sets. Target organs are eyes, heart, and arborescent organs. All organs were washed twice with cold phosphate buffer (PBS) KCl (PBS, pH 7.5) to remove blood clotting. Organs were weighed and individually homogenized with cold PBS (pH 7.5) plus cocktail inhibitor protein (Invitrogen, 1 tablet for 10 ml PBS) at a ratio of 1:10 . The homogenate is then centrifuged (Hettich) at 12,000rpm for 15 minutes at 4⁰C. The supernatant was pulled into an eppendorf (Germany) tube labeled 2ml which was safe and immediately frozen at -80⁰C. Protein quantification was carried out using the NanoDrop ND-1000 Spectrophotometer. SDS PAGE Electrophoresis uses Electrophoretic protein units (Mini Protean Tetra System BioRad). Separation gel 12% and 4% stacking gel follow the BioRad standard. Protein samples are removed from storage freezer and thawed. Protein samples were taken as much as 20μl then added with RSB in a ratio of 1: 1, put into 20ml eppendorf. With eppendorf, the sample is heated in boiling water for 5 minutes. Subsequent samples were placed in gel wells. The sample was run at 200mV for 30-30 minutes. Gel is removed and then stained with 0.1% Coomasive Blue (R, Sygma) solution for 2-4 hours. After the incubation period, the dye solution is removed and replaced with a destaining solution, incubated until the background material is completely gone. Observed protein bands formed using the Gel Doc EZ Imager. Western Blot Protein samples were removed from the freezer of -80 ° C and dithawing. Protein electrophoresed using the same procedure as SDS PAGE. The formed protein bands were transferred to membrane Nitrocellulase (Amersham Bioscience) with a Semi Dry Transfer Membrane (BioRad) system at 20V for 2 hours. Previously, the membrane and gel were soaked in the transfer buffer for 30 minutes. After the transfer process, the membrane is removed, it is confirmed that the tape has been transferred, using Ponceau Solution (Sygma). Blocking is done by dipping the membrane in 5% Carnation nonfat instant milk on 1 hour TBST in the refrigerator. Membrane was washed 0.05% TBST (Sygma) by soaking for 3 minutes while rocking slowly. Washing is repeated 3 times. The membrane is then incubated with TBX5 (Lsbio) primary antibody with a 1: 100 dilution during overnight. The membrane is washed 0.05%% TBS in the same way as above, for 3 minutes repeated 3 times. Membranes were incubated in anti rabbit-Biotin (Santa Cruz) IgG secondary antibodies in a ratio of 1: 1000 for 60 minutes. The membrane was then washed 0.05% TBT (Sygma) 3x3 minutes. Incubation with SA-HRP (dilution 1: 10,000) for 40 minutes and then washed 0.05% TBST (Sygma) 3 times 3 minutes. Final incubation using TMB-membrane substrate (KL) liquid substrate (KL) for 10 minutes. The membrane is scanned using an Epson scanner and photographed using a digital camera.