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PFKM protein (6-phosphofructokinase) is an enzyme encoded by the PFKM gene in humans. It plays a large role in the first step of glycolysis through catalysing the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate. The PFKM Polyclonal Antibody detects endogenous levels of PFKM protein.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: PFKM Polyclonal Antibody (STJ95045)
2. What was used? Primary antibody: STJ95045 PFKM Antibody Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: U87, MRC-5, HeLa cell lysates 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 at on the shaker 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: 10% 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 HeLa lysate 18ug (total protein) STJ95045 PKFM antibody 1:1000 1:5000 ~85KD 120s 2 U87 lysate 18ug(total protein) ~85KD 120s 3 MRC-5 lysate 18ug(total protein) ~85KD 120s
5. What were the results? Figure: Lane 0: Bio-Rad Precisiondual colour protein marker; Lane1: U87; Lane 2: MRC-5; Lane 3: HeLa
6. What did the customer think? Antibody Specificity: Antibody RPPA Rating: Testing results were provided independently by H. Kramer at the University of Oxford. They stated ¡°Band detected in U87 lysate approximately 45kD apparent molecular weight; expected: 85kD STJ95045 PFKM antibody¡±
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