Whole cells lysates of HCT116 and A431 were prepared according to standard procedures and the protein concentration measured with an IMPLEN NanoPhotometer P-class P360. The lysates were run on a 3–8% Bis-Tris SDS-PAGE (Life Technologies) and transferred by wet blotting (overnight, 4 °C) to a PVDF membrane (Merck/Millipore). After 1 h of blocking (PBS, 5% BSA) the membrane was cut horizontally in smaller fragments and incubated with primary antibodies over night at 4 °C. Primary antibodies used: rabbit anti-HSP90 antibody (ab13495, abcam, 1/30000), mouse anti-phospho-histone H2A.X (Ser139) antibody clone JBW301-I (Merck/Millipore, 1 μg/mL), mouse monoclonal anti-beta-actin antibody (A5316, Sigma Aldrich, 1/10000), rabbit anti-EGFR antibody [EP38Y] (ab52894, abcam, 1/5000). Next day, the membrane was rinsed in 3 times for 5 min in PBS with 1% Tween-20 and incubated with respective and anti-mouse or anti rabbit secondary antibodies with HRP-label (Life technologies). Membrane fragments were incubated with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific) and bands were visualized with a SuperCCD HR camera (Fujifilm). ImageJ (NIH) was used for quantitative analysis were relative protein levels were compared with the loading control levels from the same lysate with the same treatment.
Nanophotometer p class p360
Manufactured by Implen
Sourced in Germany
The NanoPhotometer P-class P360 is a compact and versatile UV/Vis spectrophotometer designed for accurate and reliable measurement of various samples. It features a wavelength range of 190 to 1100 nm and a path length of 0.1 mm to 10 mm, allowing for a broad range of applications. The instrument provides high-resolution photometric measurements with a wide dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Bis tris sds page, by Thermo Fisher Scientific (1 mentions)
A5316, by Merck Group (1 mentions)
Ab52894, by Abcam (1 mentions)
Ab13495, by Abcam (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Easytaq pcr supermix kit, by Transgene (1 mentions)
Peasy t3 cloning kit, by Transgene (1 mentions)
Tianpure mini plasmid kit, by Tiangen Biotech (1 mentions)
2 protocols using nanophotometer p class p360
1
Western Blot Analysis of HCT116 and A431 Cells
2
Quantitative Detection of M. paratuberculosis
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The extracted DNA of M. paratuberculosis was amplified using the 2× EasyTaq PCR Supermix kit (Beijing TransGen Biotech, China). Forward primer, 5′-ATCAGCGCGGCAC GGCTCTTG-3′, and reverse primer, 5′-CGGGTAGTTACCG CGGCGAAG-3′, were used to amplify 632 nucleotides of the IS900 gene of M. paratuberculosis (691–1322 of GenBank accession No. S74401.1; National Center for Biotechnology Information, USA). The PCR temperature profile was as follows: initial activation at 94℃ for 3 min, 35 cycles of 94℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 30 sec, and a final extension step of 72℃ for 10 min. The amplified fragment was ligated into the plasmid pEASY-T3 cloning vector by using the pEASY-T3 cloning kit (Beijing TransGen Biotech) and designated as pEASY-T3-MAP. The pEASY-T3-MAP standard DNA was extracted by using the TIANpure mini plasmid kit (Tiangen Biotech) and measured by using a NanoPhotometer P-Class P360 (Implen, Germany). The DNA copy number was calculated as described previously [19 (link)]. The DNA standard was then stored at 20℃ until needed.
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