Advansta Inc
WesternBright Sirius HRP substrate
K-12043-D10
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We are interested in understanding the regulation of protein stability of those important low abundant proteins with short half-life in tumor cells, such as Hif1alpha, Nrf2, p53, p21 and p27. One of the approaches is to detect the protein level by chemiluminescent Western blotting. We always had a difficulty in detecting those protein expressions using the ECL HRP substrate that we used to buy before. Recently, we used the WesternBright Sirius from Advansta and have easily generated clear and low background western blotting images.
Western Blot HRP Substate
Tumor cell lysates were seperated by SDS-PAGE
For western blotting, 10 ug-20ug protein lysate was separated by SDS-PAGE. The proteins were wet-transferred to 0.45um pore sized NC membrane in 100 V for 90 minutes. The membrane was blocked with 5% milk for 1 hour and incubated with primary antibody by the dilution of 1:2000 for 2hours at room temperature. WesternBlot HRP Sirius Substate was used according to the protocol provided by Advansta. Detect luminescent signal with CCD camera to adjust the exposure time.
For detection of those abundant proteins, first try dilution of primary antibody by 1:5000 to 1:10000, secondary HRP antibody shuould be diluted with more than 1:10000
When we performed western blotting to detect the low abundant proteins or phosphorylated proteins by WesternBright Sirius substrate we obtained a good blot with strong signal and low background even with longer exposure time.
None
Low background, high sensitivity and the signal lasts longer.
I can't think of any bad aspects at this moment.
Relatively low cost with best results.
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