Cell extracts were prepared with M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt™; Pierce, Rockford, IL, USA) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA) [29 (link)]. Aliquots of protein lysates were separated on SDS-8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting-grade milk (Bio-Rad, Hercules, CA, USA) in TBST (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against p16 (#92803, Cell Signaling Technology, Danvers, MA, USA), p21(sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IKK2 (GTX107970, GeneTex, Hsinchu, Taiwan) and NFκB p65 (phospho Ser536) (GTX133899, GeneTex, Taiwan), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA, USA).
Gtx133899
Manufactured by GeneTex
GTX133899 is a laboratory equipment product. It is designed to perform specific functions in a laboratory setting. The core function of this product is to facilitate scientific research and experimentation. However, a detailed and unbiased description of its features and capabilities is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Blotting grade milk, by Bio-Rad (1 mentions)
Chemiluminescence assay, by Merck Group (1 mentions)
P21 sc 6246, by Santa Cruz Biotechnology (1 mentions)
Gtx107970, by GeneTex (1 mentions)
Immobilon polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Gtx102090, by GeneTex (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Gtx104616, by GeneTex (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Las 3000, by Fujifilm (1 mentions)
2 protocols using gtx133899
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell extracts (20 μg of protein) were fractionated by 10–12% SDS–PAGE, transferred to an immobilon polyvinylidene difluoride membrane (Amersham Biosciences, Corston, UK), and probed with one of the specific primary antibodies against PTP4A3 (1:1000, GTX100600, GeneTex, Hsinchu, Taiwan), p-STAT3 (1:1000, GTX104616, GeneTex, Hsinchu, Taiwan), STAT3 (1:1000, GTX11800, GeneTex, Hsinchu, Taiwan), STAT5A (1:1000, GTX113351, GeneTex, Hsinchu, Taiwan), FOXO3A (1:1000, GTX100277, GeneTex, Hsinchu, Taiwan), TERT (1:1500, GTX124242, GeneTex, Hsinchu, Taiwan), TOP2A (1:1500, GTX100689, GeneTex, Hsinchu, Taiwan) p-JAK2 (1:1000, GTX132784, GeneTex, Hsinchu, Taiwan), JAK2 (1:1000, GTX01195, GeneTex, Hsinchu, Taiwan), p-P65 (1:1000, GTX133899, GeneTex, Hsinchu, Taiwan), P65 (1:1000, GTX102090, GeneTex, Hsinchu, Taiwan) and β-actin (1:10,000, #4970, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Subsequently, the membrane was hybridized with appropriate HRP–conjugated secondary antibodies for 1 h at room temperature. Finally, immune complexes were visualized using the chemiluminescence method with an ECL detection kit (Merck Millipore, Temecula, CA, USA) on Fujifilm LAS-3000 (Fujifilm, Tokyo, Japan).
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!