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Fukutin-related protein (FKRP) is targeted to the medial Golgi apparatus and is necessary for post-translational modification of dystroglycan. Mutations in the gene encoding this protein have been associated with congenital muscular dystrophy, mental retardation, and cerebellar cysts. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.
1. HOW DID OUR ANTIBODY PERFORM? CUSTOMER REVIEW: FKRP Polyclonal Antibody (STJ93080)
2. WHAT WAS USED? Primary antibody: STJ93080 FKRP Polyclonal Antibody Provider: St John’s Laboratory Dilution ratio: 1:500 Application: Western Blot. Materials for validation: HeLa cell line
3. WHAT WAS THE PROTOCOL? Treatment of materials: When cells were cultured to 90% plating density, RIPA buffer with protease inhibitors (Roche) and equal volume of 2X loading buffer were added for 10 min water bath at 100?C. Gel electrophoresis: 4-20% polyacrylamide gel (Bio-Rad), constant voltage 180V, 40 min. Transfer: 0.25 nm nitrocellulose membrane, by using the Trans-Blot Turbo transfer system (Bio-Rad) for 7 minutes. Blocking: 1X TBST with 5% skimmed milk powder for 1 hr at room temperature. Primary antibody probing: Primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4?C overnight. Membrane wash: 1X TBST wash for 3 times. Secondary antibody probing: Secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1:10000 dilution ratio, for 1 hr. Membrane wash: 1X TBST wash for 3 times. Visualization: Chemiluminescent signal obtained by using the SuperSignal West Chemiluminescent Substrate (Pierce) were detected using the ChemiDoc? XRS system (BioRad) with an exposure time of 2 minutes.
4. WHAT WAS THE PROTOCOL? Gel electrophoresis information£º4-20% Mini-Protean TGX Gels (Bio-Rad), constant voltage 180 V, 40 min. Transfer information£º0.25 nm nitrocellulose membrane, by using the Trans-Blot Turbo transfer system (Bio-Rad) for 7 minutes. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band (KDa) Visualization time 1 HeLa lysate 20 ug STJ93080. FKRP Polyclonal Antibody 1:500 1:10000 ~55KDa 2 mins
5. WHAT DID THE CUSTOMER THINK? Testing results were provided by Manuela Lanzafame, PhD from Istituto di Genetica Molecolare-CNR, Italy. They stated that ¡°In HeLa cells the STJ93080 FKRP antibody detects a band of the predicted molecular weight of ~55 KDa and two additional bands.¡±
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