The cells were lysed in RIPA lysis buffer (Millipore) containing protease (Bimake) and phosphatase (Bimake) inhibitors. The samples were resolved by SDS-PAGE and transferred to PVDF membrane after blocking, the membranes were incubated with primary antibodies overnight at 4 °C and then exposed to secondary antibodies. Protein bands were visualized using West Femto Substrate Trial Kit (Thermo Scientific). The primary antibodies included anti-p38 MAPK (Cell Signaling Technology), anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), anti-STAT3 (Abcam), anti-phospho-STAT3 (Tyr705) (Abcam), anti-LC3B (Sigma), anti-Beclin1 (proteintech), anti-β-Tubulin (Cell Signaling Technology), and anti-GAPDH (proteintech). The secondary antibodies were goat anti-mouse IgG HRP (proteintech) and goat anti-rabbit IgG HRP (proteintech).
West femto substrate trial kit
Manufactured by Thermo Fisher Scientific
The West Femto Substrate Trial Kit is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed for the detection of low-abundance proteins in Western blot analysis. The kit provides a trial-sized quantity of the West Femto Maximum Sensitivity Substrate, which can be used to generate chemiluminescent signals for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3b, by Merck Group (1 mentions)
Hrp goat anti rabbit igg, by Proteintech (1 mentions)
Anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Anti beclin 1, by Proteintech (1 mentions)
Goat anti mouse igg hrp, by Proteintech (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Anti p38 mapk, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti phospho stat3 tyr705, by Abcam (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti stat3, by Abcam (1 mentions)
Sds page loading buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti ubiquitin antibody, by Merck Group (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
2 protocols using west femto substrate trial kit
1
Protein Expression and Activation Analysis
2
Proteomic Analysis of Bortezomib-Treated 4T1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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UPs were enriched from 4T1 tumor cell lysate treated with bortezomib and ammonium chloride by α-Al2O3-CONH-Vx3, and the whole cell lysate, unbound lysate (the supernatant after the centrifugation for α-Al2O3-UPs collection from the whole cell lysate) and α-Al2O3-UPs were collected for Western blot analysis. All the samples were mixed with SDS-PAGE loading buffer (Invitrogen) and boiled for 5 mins. Then, the samples were centrifuged at 12000 g for 10 mins and the supernatant was resolved by 12.5% SDS-PAGE (Invitrogen). Then, the proteins were transferred to a PVDF membrane, blocked by 5% dry milk for 1 hr, incubated with primary antibody overnight at 4°C, and exposed to HRP-conjugated secondary antibody for 1 hr. The membrane was revealed using West Femto Substrate Trial Kit (Thermo Fisher). The primary antibody was anti-ubiquitin antibody (1:1000, Sigma, #3933) and the secondary antibody was goat anti-rabbit IgG HRP (1:5000, eBioscience).
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