Proteins were extracted from pancreatic adenocarcinoma cells using RIPA lysis buffer (CWBIO, Beijing, China) containing phosphatase inhibitors and protease inhibitors. The protein concentration was determined using BCA reagent (CWBIO). Protein samples were separated by 8%‒10% SDS-PAGE, and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA). Then, PVDF membrane was blocked in 5% skim milk and incubated with mouse anti-NQO1 (1:1000; Abcam, Cambridge, USA), rabbit anti-E-cadherin (1:1000; Abcam), rabbit anti-Vimentin (1:1000; Abcam), rabbit anti-Snail (1:1000; Abcam), rabbit anti-CPT1A (1:1000; Abcam), rabbit anti-LCAD (1:1000; Proteintech, Chicago, USA), rabbit anti-MCAD (1:1000; Proteintech), or mouse anti-actin (1:3000; Santa Cruz Biotech, Santa Cruz, USA) primary antibody in a refrigerator at 4°C overnight, followed by incubation with corresponding HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Beyotime, Shanghai, China) at room temperature for 1-2 h. Finally, an enhanced chemiluminescence (ECL) kit (CWBIO) was used for the visualization of the blots.
Rabbit anti cpt1a
Manufactured by Abcam
Sourced in United States
Rabbit anti-CPT1a is a primary antibody that specifically recognizes the carnitine palmitoyltransferase 1A (CPT1A) protein. CPT1A is an enzyme involved in the transport of long-chain fatty acids into the mitochondria for beta-oxidation. This antibody can be used for the detection and analysis of CPT1A in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti vimentin, by Abcam (1 mentions)
Mouse anti actin, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by CWBIO (1 mentions)
Bca reagent, by CWBIO (1 mentions)
Mouse anti nqo1, by Abcam (1 mentions)
Rabbit anti snail, by Abcam (1 mentions)
Rabbit anti e cadherin, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Mouse anti p53, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Insulin syringe, by BD (1 mentions)
Goat anti rabbit alexa568, by Thermo Fisher Scientific (1 mentions)
Mouse anti ncl, by Santa Cruz Biotechnology (1 mentions)
Tritc labeled phalloidin, by Merck Group (1 mentions)
P6148, by Merck Group (1 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti pfkfb3, by Abcam (1 mentions)
8 well chamber slide, by Merck Group (1 mentions)
Rabbit anti hk2, by Proteintech (1 mentions)
3 protocols using rabbit anti cpt1a
1
Protein Expression Analysis in Pancreatic Cancer
2
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA buffer and mechanical disruption through a 1ml insulin syringe (BD).
Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V and transferred to a nitrocellulose membrane at 100 V for 1.5 hours. Unspecific binding sites were blocked with 5% milk in TBS-Tween 0.05% for 30 minutes. The membrane was probed with mouse anti-Nucleolin (NCL, 1:500, Santa Cruz), rabbit anti-Hexokinase-2 (HK2, 1:500, proteintech), rabbit anti-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3, 1:1,000, abcam), mouse anti-p53 (1:1,000, Cell signalling), rabbit anti-CPT1a (1:500, abcam), and rabbit anti-TIGAR (1:200, Sigma) at 4°C overnight. A secondary donkey antibody directed against mouse, or rabbit (1:2,000, Dako) was applied for 1 hour. Bands were visualised by chemiluminescence using ECL (Amersham). Densitometric analysis was performed with ImageJ (NIH freeware). Data were normalised to actin, and values of control cells were set to 1.
Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V and transferred to a nitrocellulose membrane at 100 V for 1.5 hours. Unspecific binding sites were blocked with 5% milk in TBS-Tween 0.05% for 30 minutes. The membrane was probed with mouse anti-Nucleolin (NCL, 1:500, Santa Cruz), rabbit anti-Hexokinase-2 (HK2, 1:500, proteintech), rabbit anti-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3, 1:1,000, abcam), mouse anti-p53 (1:1,000, Cell signalling), rabbit anti-CPT1a (1:500, abcam), and rabbit anti-TIGAR (1:200, Sigma) at 4°C overnight. A secondary donkey antibody directed against mouse, or rabbit (1:2,000, Dako) was applied for 1 hour. Bands were visualised by chemiluminescence using ECL (Amersham). Densitometric analysis was performed with ImageJ (NIH freeware). Data were normalised to actin, and values of control cells were set to 1.
3
Immunofluorescence Staining of HUVECs
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Cells were cultured on nanopillars or 8-well chamber slides (Sigma-Aldrich), cells were fixed with 4% PFA (P6148, Sigma-Aldrich) for 15 min at room temperature (RT). The cell membrane was then permeabilized with 0.1% Triton X-100 (X100, Sigma-Aldrich) in PBS (10 min) followed by 1% BSA/PBS (85040C, Sigma-Aldrich) blocking step (30 min, RT).
HUVECs were stained with with mouse anti-NCL (1:100, Santa Cruz), rabbit anti-HK2 (1:100, proteintech), rabbit anti-PFKFB3 (1:100, abcam, rabbit anti-CPT1a (1:500, abcam), and rabbit andti-TIGAR (1:200, Sigma) in 1% BSA/PBS at 4°C overnight. Cells were the incubated with secondary antibodies goat anti-rabbit Alexa 568 and goat anti-mouse Alexa 488 (1:500, Thermo Fisher Scientific), or TRITC-labeled phalloidin (1:100 Sigma) in 1% BSA/PBS for 1.5 hours at RT. Nuclei were counterstained with DAPI staining using (1:20,000, Thermo Fisher Scientific) in PBS for 5 min.
HUVECs were stained with with mouse anti-NCL (1:100, Santa Cruz), rabbit anti-HK2 (1:100, proteintech), rabbit anti-PFKFB3 (1:100, abcam, rabbit anti-CPT1a (1:500, abcam), and rabbit andti-TIGAR (1:200, Sigma) in 1% BSA/PBS at 4°C overnight. Cells were the incubated with secondary antibodies goat anti-rabbit Alexa 568 and goat anti-mouse Alexa 488 (1:500, Thermo Fisher Scientific), or TRITC-labeled phalloidin (1:100 Sigma) in 1% BSA/PBS for 1.5 hours at RT. Nuclei were counterstained with DAPI staining using (1:20,000, Thermo Fisher Scientific) in PBS for 5 min.
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