The indicated cells were lysed in RIPA buffer on ice for 30 min and cell debris were discarded via refrigerated centrifugation (12,000 rpm for 15 min). Equal amount of cell lysates was resolved by SDS-PAGE and then transferred to PVDF membrane in ice bath. The membrane was blocked with 5% milk in TBST buffer (0.05% Tween-20), and incubated with primary antibody at 4 °C overnight. After wash with TBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 h. The protein bands were then visualized with enhanced chemiluminescence reagent (Cwbiotech).
Enhanced chemiluminescence reagent
Manufactured by CWBIO
Sourced in China
Enhanced chemiluminescence reagent is a laboratory reagent used for the detection and quantification of proteins in Western blot analysis. It provides a sensitive and reliable method for visualizing and measuring protein levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti p akt, by Proteintech (1 mentions)
Anti p mtor, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf member, by Merck Group (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti crabp2, by Proteintech (1 mentions)
Bca kit, by CWBIO (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Anti bax, by Proteintech (1 mentions)
Anti mtor, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sc 56 770, by Santa Cruz Biotechnology (1 mentions)
Zb 2305, by OriGene (1 mentions)
Ab109394, by Abcam (1 mentions)
Ab40866, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Ab8106, by Abcam (1 mentions)
6 protocols using enhanced chemiluminescence reagent
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis of Dezocine
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RIPA Lysis Buffer (CWBIO, Beijing, China) was used to extract the proteins in cells treated with dezocine for 48 h. Following which the concentration was determined by BCA kit (CWBIO), the protein samples (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF members (Millipore, Billerica, MA, USA). After being blocked with 5% dried skimmed milk for 1 h, the members were incubated with primary antibodies at 4°C overnight, and then incubated with HRP-conjugated secondary antibodies at room temperature. An enhanced chemiluminescence reagent (CWBIO) was performed to visualize the blot bands. The antibodies, including anti-Bcl-2 (Cat no. 12789-1-AP), anti-Bax (Cat no. 50599-2-Ig), anti-p-Akt (Cat no. 66444-1-Ig), anti-mTOR (Cat no. 20657-1-AP), anti-GAPDH (Cat no. 10494-1-AP), and anti-CRABP2 (Cat no. 10225-1-AP) were obtained from Proteintech Group (IL, USA); anti-cleaved Caspase 3 (Cat no. 9661), anti-Akt (Cat no. 9272), anti-p-mTOR (Cat no. 5536), anti-p70s6k (Cat no. 9204), and secondary antibodies were obtained from Cell Signaling Technology (Danvers, USA).
3
Western Blot Protein Analysis
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The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in TBST with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C followed by incubation with rabbit secondary antibodies for 1 h. Antibody binding was detected on autoradiographic film using the enhanced chemiluminescence reagent (CWBIO, Beijing, China).
4
Immunoblotting Analysis of DNA Damage Response
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Cells were washed with prechilled PBS and harvested into lysis buffer (150 mM NaCl, 10% glycerol, 0.3% Triton X-100, 50 mM Tris pH 8.0). Proteins were resolved via SDS-PAGE and transferred to PVDF membrane. Immunoblotting was performed with the following antibodies anti-MTA2 (ab8106, Abcam, Cambridge, UK, 1:1000), anti-CHK1 (ab40866, Abcam, Cambridge, UK, 1:200), anti-CHK1 pS345 (#2348, Cell Signaling Technology, MA, USA, 1:1000), anti-CHK1 pS317 (ab59239, Abcam, Cambridge, UK, 1:1000), anti-RPA32 (sc-56,770, Santa Cruz, CA, USA, 1:1000), anti-RPA32 pT21 (ab109394, Abcam, Cambridge, UK, 1:5000), anti-γH2AX (sc-517,348, Santa Cruz, CA, USA, 1:1000), anti-β-actin (#3700, Cell Signaling Technology, MA, USA, 1:1000), anti-rabbit IgG, HRP-linked (ZB-2301, ORIGENE, China, 1:10,000), anti-mouse IgG, HRP-linked (ZB-2305, ORIGENE, China, 1:10,000), and detected using enhanced chemiluminescence reagent (CWBIO, Beijing, China).
5
Quantitative Western Blot Analysis
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Western blot was performed according to standard protocols as described previously.13,14 (link) To put it briefly, cells were collected and lysed with RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, Beyotime) containing 1% protease inhibitor. Then lysates were centrifuged at 12 000g for 15 min at 4 °C, and the supernatants were collected for further use. Thirty micrograms (30 μg) of proteins were denatured in loading buffer and loaded on a gel after which SDS-PAGE was performed. Resolved bands were transferred to nitrocellulose membranes which were incubated with anti-CS, anti-EPRS and GAPDH respectively at 4 °C overnight followed by incubation with HRP-labeled goat anti-rabbit secondary antibodies. Finally, the bands were visualized using enhanced chemiluminescence reagent (CWBIO, China). The grey values of these target bands were measured using Image J software and histograms were plotted using GraphPad Prism software 7.04 (GraphPad Prism software, Inc. San Diego, USA).
6
Western Blot Analysis of Metabolic Enzymes
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Western blot analysis was performed according to protocols described recently 6 . Briefly, cells from different groups were lysed using RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, Beyotime, China) containing 1% protease inhibitor. Lysates were centrifuged for 15 min at 4 °C (12000 g), after which the supernatant was collected for further use. Proteins concentration were quantified with the Broadford assay kit (CWBIO, China). Equal amounts (20 µg) of proteins were denatured by heating and separated by SDS-PAGE, followed by transfer to nitrocellulose membranes which were later incubated with designated primary antibodies against PKM (Abcam, #38237, UK), GLS2 (Abcam, #113509, UK), GLUL (Abcam, #49873, UK), LDHA (Abcam, #125683, UK) and β-actin (Cell Signaling, #5176, USA) (dilution 1:1000) respectively at 4 °C overnight after blocking with 5% skimmed milk. Thereafter, membranes were incubated with suitable secondary antibodies (ZSGB-Bio, China) (dilution 1:3000) at room temperature. Finally, chemiluminescence signals were visualized using an enhanced chemiluminescence reagent (CWBIO, China). The grey values of these signals were measured using Image J and histograms were plotted using GraphPad Prism 8.0.1.
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