Proteins were subjected to 12.5% discontinuous SDS-PAGE, separated by electrophoresis and electroblotted to PVDF membranes (Milipore Corp., Billerica, MA, USA). Specific antibodies against NFkB (#3033, 1:1000 in 5%-BSA-TBST, Cell Signalling) were used to detect bands at 65 kDa. For the corresponding loading control, an antibody against GAPDH was used (gt239, 1:2000, Genetex Inc.). Primary antibodies were incubated at 4°C over night, the according HRP-Secondary antibodies were incubated for one hour at room temperature and binding was detected with a chemiluminescence-based detection kit (Immobilon Western, Milipore Corp.).
Gt239
Manufactured by GeneTex
The GT239 is a high-performance reagent for use in nucleic acid amplification techniques, such as real-time PCR. It is designed to provide reliable and consistent results in gene expression analysis and other molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Bsa tbst, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Oligonucleotides, by Cosmogenetech (1 mentions)
Ac 15, by Merck Group (1 mentions)
Mitochondrial marker antibody sampler kit, by Cell Signaling Technology (1 mentions)
3 protocols using gt239
1
Western Blot Analysis of NF-kB
2
Antibody-based Protein Detection Techniques
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For primary antibodies, we used mouse antibody against PARP1 (cat# 556494, 1 : 1,000, BD Bioscience), mitochondrial marker antibody sampler kit (cat# 8674, 1 : 3,000, Cell Signaling, rabbit antibody to CoxIV, Cytochrome C, HSP60, PHB1, pyruvate dehydrogenase, SDHA, and mouse antibody to SOD1), mouse antibody against GAPDH (GT239, 1 : 5,000, GeneTex), mouse antibody against FLAG (clone M2, Sigma), rabbit antibody against PINK1 (cat# 100-494, 1 : 2,000, Novus Biologicals), and mouse antibody against UQCRC2 (cat# ab14745, 1 : 3,000, Abcam). For secondary antibodies, we used horseradish peroxidase- (HRP-) conjugated mouse antibody against FLAG (cat# A8592, 1 : 5,000, Sigma-Aldrich), HRP-conjugated antibody against HA (1 : 3,000, Roche), HRP-conjugated mouse antibody against β-actin (AC15, 1 : 10,000, Sigma-Aldrich), HRP-conjugated sheep antibody against mouse IgG (cat# RPN4301, 1 : 5,000, GE Healthcare), HRP-conjugated donkey antibody against rabbit IgG (cat# RPN4101, 1 : 5,000, GE Healthcare), Alexa Fluor 488-conjugated donkey antibody against mouse IgG (H+L) (cat# A21202, 1 : 1,000, Invitrogen), and Alexa Fluor 568-conjugated donkey antibody against rabbit IgG (cat# A10042, 1 : 1,000, Invitrogen). LentiCRISPR and lentiCRISPR-sgRNA-eGFP#2 constructs were purchased from Addgene. Oligonucleotides were ordered from and synthesized by Cosmo Genetech.
3
Temporal Regulation of Creb3 Factors
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Mice were fasted for 24 hours and refed with standard chow diet plus 20% glucose solution for 0, 0.5, 2 or 4 hours. The sacrifice was always performed at noon in all groups to avoid circadian effects. Livers collected were snap-frozen and stored at -80ºC. RNA from 15mg of mice liver was extracted and cDNA prepared as described above. RTqPCR was done using primers amplifying small regions of Creb3L1 and Creb3L2. Data was normalized to the house-keeping gene TATA-binding protein (TBP). For westerns, whole-cell protein lysates were prepared from 20mg of liver and analyzed as described above.
Mouse monoclonal anti-Creb3L2 (Merck, MABE1018) and anti-GAPDH (Genetex, GT239) antibodies were used.
Mouse monoclonal anti-Creb3L2 (Merck, MABE1018) and anti-GAPDH (Genetex, GT239) antibodies were used.
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