Anti-Creatine kinase B type/CKB Antibody Picoband®

Western blot analysis of CKB using anti-CKB antibody (A01695-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: human placenta tissue lysates,
Lane 3: human COLO-320 whole cell lysates,
Lane 4: human SW620 whole cell lysates,
Lane 5: human MDA-MB-231 whole cell lysates,
Lane 6: rat brain tissue lysates,
Lane 7: mouse brain tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CKB antigen affinity purified polyclonal antibody (Catalog # A01695-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CKB at approximately 45KD. The expected band size for CKB is at 43KD.

Immunohistochemistry of CKB and MTCK in CRCs. ( A , B ) Immunohistochemical examination of CKB (A) and MTCK (B). Immunohistochemical activity is classified into grade 0–3. Grade 0 is equivalent to the expression in the normal colonic epithelium. ( C ) Histogram of expression grade. ( D ) Immunohistochemical examination of CKB and MTCK in case of well-differentiated adenocarcinoma (tub1), signet ring cell carcinoma (sig), primary tumor and liver metastasis of tub1 case. Scale bar, 100 μm. Abbreviations: CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; CRC: colorectal cancer.
Index in PubMed under a CC BY license. PMID: 37204253

Creatine kinase inhibitory effect of DNFB. ( A ) Protein fractionation of mitochondria (Mitochondrial) and mitochondria-free cytosol (Cytosol). MTCK and CKB were detected by western blotting. TOM20 and GAPDH were subjected as a mitochondrial marker and a cytosol marker, respectively. ( B ) Inhibition of creatine kinase activity (at 20 min) of CKB (cytosol fraction) and MTCK (mitochondrial fraction) in a DNFB concentration-dependent manner. ( C , D ) Inhibition of creatine kinase activity of CKB (C) and MTCK (D) in a time-dependent manner. Error bar, standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; DNFB: dinitrofluorobenzene; TOM20: translocase of the outer membrane 20; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
Index in PubMed under a CC BY license. PMID: 37204253

Effect of creatine shuttle inhibition by DNFB on growth and stemness of CRC cells. ( A ) Effect of DNFB on cell growth. ( B ) Effect of pCr on DNFB-treated CRC cells. ( C ) Effect of DNFB (10 μM) on expression of stemness-associated genes detected by quantitative RT-PCR. β-Actin was used as a loading control. ( D ) Effect of DNFB (10 μM) on sphere formation. Scale bar, 50 μm. ( E , F ) Effect of knockdown of CKB or MTCK on cell proliferation (E) and sphere formation (F). Error bars: standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; DNFB: dinitrofluorobenzene; CRC: colorectal cancer; OD: optimal density; pCr: phosphocreatine; C: untreated control.
Index in PubMed under a CC BY license. PMID: 37204253

Creatine shuttle provides ATP for EGFR phosphorylation in EGF-treated HT29 cells. ( A ) Effect of DNFB (5 μM) and knockdown of MTCK or CKB on EGFR phosphorylation. Lower panels, effect of siMTCK and siCKB on expression of MTCK and CKB, respectively. ( B ) Semi-quantification of EGFR phosphorylation levels. ( C ) Effect of ATP on suppressed EGFR phosphorylation by DNFB (5 μM). ( D ) Effect of oligomycin on EGFR phosphorylation. ( E ) Effect of CyCr on creatine phosphorylation. ( F ) Effect of CyCr on EGFR phosphorylation. ( G ) Co-immunoprecipitation using anti-CKB antibody or anti-EGFR antibody to examine binding of CKB and EGFR. ( H ) Duolink ® proximity ligation assay. Red, proximity signal of EGFR and CKB. Blue, DAPI. Scale bar, 10 μm. Error bars: standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: DNFB: dinitrofluorobenzene; EGFR: epithelial growth factor receptor; pEGFR: phosphorylated EGFR; EGF: epithelial growth factor; CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; siC: short interfering RNA (siRNA) used as control; siMTCK: siRNA for MTCK; siCKB: siRNA for CKB; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; OM: oligomycin; CyCr: cyclocreatine; pCr: phosphocreatine; IP: immunoprecipitation; CB: Coomassie blue; WCL: whole cell lysate; DAPI: 4′,6-diamidino-2-phenylindole.
Index in PubMed under a CC BY license. PMID: 37204253

IHC analysis of CKB using anti-CKB antibody (A01695-1).
CKB was detected in paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CKB Antibody (A01695-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CKB using anti-CKB antibody (A01695-1).
CKB was detected in paraffin-embedded section of mouse kidney tissue . Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CKB Antibody (A01695-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CKB using anti-CKB antibody (A01695-1).
CKB was detected in paraffin-embedded section of rat kidney tissue . Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CKB Antibody (A01695-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.