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UCH-L1 (Ubiquitin carboxyl-terminal hydrolase L1) is a cysteine protease and is highly abundant in neuronal cells in the brain. It plays a significant part in protein degradation through its hydrolytic activity in which it cleaves a peptide bond at the ubiquitin glycine C-terminal in order to recycle ubiquitin from already degraded proteins.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: UCH-L1 Polyclonal Antibody (STJ96177)
2. What was used? Primary antibody: STJ96177 UCH-L1 Antibody Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: U87 cell lysate, MRC-5 cell lysate, UCH-L1 protein as positive control Click for product data sheet PDF
3. What Was The Protocol? Treatment of Materials Cell pellets were lysed in RIPA lysis buffer with protease inhibitor cocktail ( Roche) and equal volume of 2X loading buffer (Sigma) were heated at 95?C for 10 minutes in a heat block. Gel Electrophoresis 12% Polyacrylamide gel with a constant voltage of 185V for 60 minutes. Transfer 0.45 um NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 minutes. Blocking 1X TBST with 5% skimmed milk powder was used and left overnight. Primary Antibody Probing Primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder and incubated on the shaker at room temperature for 1 hour. Membrane Wash Membrane was washed 3 times with 1X TBST, with a 5 minute incubation on the shaker in between each wash. Secondary Antibody Probing secondary antibody was added to the solution with a 1:5000 dilution ratio with mouse mAb for a blue ladder (dilution 1:2500) for 1 hour. Membrane Wash Membrane was washed 3 times with 1X TBST, with a 5 minute incubation on the shaker in between each wash, then finally washed with deionized water. Visualization ECL visualization for 5 minutes (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).
4. What was the protocol? Gel electrophoresis information: 12% polyacrylamide gel, constant voltage of 185V for 60 minutes. Transfer information: 0.45 um NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 minutes. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 U87 cell lysate 18ug(total protein) STJ96177. UCH-L1 antibody 1:1000 1:5000 ~28KD 1s 2 MRC-5 cell lysate 18ug(total protein) STJ96177. UCH-L1 antibody 1:1000 1:5000 ~28KD 1s 3 UCH-L1 protein 50ng (purified protein) STJ96177. UCH-L1 antibody 1:1000 1:5000 ~28KD 1s
5. What were the results?Marker 1 2 3 Anti-Actin Figure: Lane1: U87; Lane 2: MRC-5, Lane 3: UCH-L1 Recombinant protein
6. What did the customer think? Antibody Specificity: Antibody Rating: Testing results were provided independently by H. Kramer at the University of Oxford. They stated ¡°In U87, MRC-5 cell lysates and UCH-L1 recombinant protein, there were strong target bands detected by UCH-L1 antibody STJ96177.¡±
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