Cells were lysed in a reducing NuPAGE sample loading buffer (Invitrogen). Equal amounts of proteins were separated onto precast gels (NuPAGE, Invitrogen) and transferred to Hybond TM nitrocellulose membrane obtained from GE Healthcare (Pittsburgh, Pennsylvania, USA). Blots were blocked in Tris buffered saline with 0, 1% Tween-20 (TBST) and 5% nonfat dry milk and incubated overnight with the appropriate primary antibody. The following primary antibodies and dilutions were used: human p53 from Santa Cruz Biotechnology (Dallas, Texas, USA), sc-6243 1:500; human p21 (Santa Cruz Technology, sc-6246 1:500); human MDM2 (Santa Cruz Biotechnology, sc-965 1:500); human SCD from Cell Signaling (Beverly, Massachusetts, USA), M38 1:1000); human SREBP1 from Active Motif (Carlsbad, California, USA), 39939 1:1000); anti AKT/PKB (Invitrogen, 44–609G 1:1000), Phospho-AKT (Ser473), (Invitrogen, 44–621G 1:1000) and GAPDH (Invitrogen 1:10000). Immunoreactive proteins were detected with the Enhanced Chemiluminescence Western Blotting Detection System from Amersham Biosciences (Pittsburgh, Pennsylvania, USA).
Hybond tm
Manufactured by GE Healthcare
Sourced in United States
Hybond TM is a series of nylon-based transfer membranes designed for various blotting applications, including Northern, Southern, and Western blotting. The membranes are optimized for the efficient capture and immobilization of nucleic acids or proteins from electrophoretic gels. Hybond TM membranes provide reliable performance and consistent results in a range of blotting protocols.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blotting detection system, by Cytiva (1 mentions)
Human p21, by Santa Cruz Biotechnology (1 mentions)
Human p53, by Santa Cruz Biotechnology (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Nupage sample loading buffer, by Thermo Fisher Scientific (1 mentions)
Phospho akt ser473, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Anti myc antibody, by Santa Cruz Biotechnology (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
2 protocols using hybond tm
1
Protein Expression Analysis by Western Blot
2
Fluorescence Resonance Energy Transfer Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
[γ-32P] ATP was purchased from Perkin-Elmer (Waltham, MA, USA), SuperSignal West Pico Chemiluminescent substrate kit from Thermo Fisher Scientific Inc. (Waltham, MA, USA) and polyvinylidene difluoride membranes (HybondTM) from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Horseradish peroxidase (HRP)-conjugated anti-glutathione S-transferase (GST) antibodies were purchased from Abcam (Cambridge, MA, USA), anti-phospho-PAK antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA), anti-total myelin basic protein (MBP) antibodies from Millipore (Billerica, MA, USA) and anti-myc antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Plasmids to express the YFP-PBD and CFP-V12Cdc42 fusion proteins for fluorescence resonance energy transfer (FRET) analysis were a gift from Dr Michiyuki Matsuda.13 (link)
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!