LoVo cells protein extracts were prepared using whole-cell lysis buffer (50 mM HEPES, 1 mM EGTA, 150 mM NaCl, 1.5 mM MgCl 2 , 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, and 1% Triton X-100) containing a protease inhibitor (Sigma-Aldrich). A total of 30 mg of protein was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 12% gel and transferred electrophoretically onto nitrocellulose blotting membranes (Pall Corporation). The membranes were blocked in 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 hour. The membranes were incubated overnight at 4 C with specific primary antibodies against PLAC8 (A62753; Sigma-Aldrich) and b-actin (TA-09; Zhongshan Golden Bridge). Then the membranes were incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 1.5 hours. Immunodetection was performed with a Super Signal West Femto Trial Kit (Thermo Fisher Scientific), and images were acquired using the Gene Gnome Imaging System (Syngene Bio-imaging).
Nitrocellulose blotting membrane
Manufactured by Pall Corporation
Sourced in Mexico, United States
Nitrocellulose blotting membranes are a type of laboratory equipment used in various analytical techniques. They serve as a support medium for the transfer and immobilization of biomolecules, such as proteins, nucleic acids, or carbohydrates, for further analysis or detection.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Odyssey imager, by LI COR (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
A62753, by Merck Group (1 mentions)
Supersignal west femto trial kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Genegnome imaging system, by Syngene (1 mentions)
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Anti pak5, by Abcam (1 mentions)
Anti β actin, by ZSGB-BIO (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Ibrighttm fl1000 imaging system, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
9 protocols using nitrocellulose blotting membrane
1
Western Blot Analysis of PLAC8 Protein
2
Protein Quantification and Western Blot Analysis
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, People’s Republic of China) and quantified by Enhanced BCA Protein Assay Kit (Beyotime). Sample proteins were resolved in a SDS-PAGE loading buffer. Equivalent protein from each sample was separated by SDS-PAGE electrophoresis and transferred to nitrocellulose blotting membranes (Pall Corporation, Mexico). Then, the membranes were blocked and incubated with specific primary antibodies: Sp1 (Abcam), p300 (Abcam), Ki-67 (Abcam), β-Actin (ZSGB-BIO, Beijing, People’s Republic of China) for overnight at 4°C. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Vicmed) for 2 hrs. Protein bands were determined using the TanonTM High-sig ECL Western Blot Substrate (Tanon, Shanghai, People’s Republic of China) and analyzed by Image analysis software (Tanon). For Co-IP, protease inhibitor cocktail (Sigma Aldrich, MO, USA) was added in the cell lysates. Then, cell lysates for IP were incubated with corresponding antibodies overnight at 4°C. Subsequently, protein A/G beads (Santa Cruz, Shanghai, People’s Republic of China) were added, and the cell lysates were mixed by rotation. Finally, immunoprecipitated proteins were detected by SDS-PAGE as performed before.
3
Western Blot Analysis of PAK5 Protein
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Total protein was distilled using RIPA lysis buffer (Beyotime, Shanghai, China) and thereby gauged by Enhanced BCA Protein Assay Kit (Beyotime). Equivalent proteins from each sample were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose blotting membranes (Pall Corporation, Mexico). Subsequently, the membranes were blocked with tris-buffered saline containing Tween-20 (TBST, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.05% Tween-20) and 5% non-fat dry milk for 2 h at room temperature (r/t) and incubated with specific primary antibodies: anti-PAK5 (1: 500, Abcam, Shanghai, China, catalog: ab110069) or anti-β-actin (1: 1000, ZSGB-BIO, Beijing, China, catalog: TA-09) for overnight at 4 °C. Having been rinsed thrice, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000, Vicmed, catalog: VA001 and VA002) for 2 h at r/t. Protein bands were determined using a Tanon High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) and analyzed by Image analysis software (Tanon). The grey levels of western blot analysis were measured and quantified by ImageJ software. The grey levels of PAK5 were normalized to those of β-actin and expressed as a percentage of control. All experiments were performed in triplicate.
4
Immunoblotting Protein Extraction and Analysis
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For immunoblotting, cells were harvested with ice-cold modified radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), 100 mM potassium chloride, 80 mM sodium fluoride, 10 mM ethylene glycol tetraacetic acid, 50 mM h-glycerophosphate, 10 mM p-nitrophenyl phosphate, 1 mM vanadate, 0.5% sodium deoxycholate, and 1% NP40. Protein concentrations were determined with using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, United States). Equal amounts of protein lysates were separated by SDS-PAGE and then transferred to nitrocellulose blotting membranes (Pall Corporation, Port Washington, NY, United States). Immunoblots were blocked, incubated with the primary antibody, washed, and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, United States), and visualized using western blotting luminol reagent (Santa Cruz Biotechnology). Western blot bands were imaged and quantified using the iBrightTM FL1000 Imaging System (Thermo Fisher Scientific).
5
Western Blot Analysis of Recombinant Cap Proteins
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Western blot analysis was performed following the established procedures. After electrophoresis, the purified rCap proteins and pET-32a vector proteins as controls were electrotransferred onto nitrocellulose blotting membranes (Pall Corporation, USA). After blocking the membranes by using 5% skimmed milk-PBS and washing three times with PBS-T (0.05% Tween 20 dissolved in PBS), the membranes were incubated for 2 h at 37°C with serum samples diluted in 1:100. The membranes were washed three times with PBS-T, and further incubated for 1 h with horseradish peroxidase-conjugated rabbit anti-dog IgG (Bioss antibodies, Beijing, China) at a dilution of 1:5,000. After washing three times, the bands were detected using DAB reagents (Biotopped, Beijing, China).
6
Western Blot Protein Quantification
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Total protein was extracted from tissues or cells using lysis buffer containing protease inhibitors. The extracted proteins were quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose blotting membrane (PALL Corporation, Port Washington, NY). The membranes were blocked for 1 h in 5% skim milk in 1 × Tris-buffered saline Tween and incubated overnight with primary antibodies at 4°C. Last, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Fluorophore-conjugated antibodies were detected using the Odyssey Imager (LI-COR Biosciences Inc., Lincoln, NE). The primary antibodies used were mouse anti-Rnf217 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti–β-actin (1:2000, KangChen Bio-tech, Shanghai, China). The secondary antibodies were IRDye 680-conjugated goat anti-rabbit (Abcam, Cambridge, Cambridgeshire, UK) and IRDye 800CW-conjugated goat anti-mouse (Abcam).
7
Western Blot Analysis of Cyclin D2 Protein
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Total protein extracts were prepared using cell lysis buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, 1 mM pepstatin, 80 mM aprotinin). Protein contents were quantified by the Bradford reagent according to the manufacturer’s instructions. Equal amounts of proteins were separated by 12% SDS-PAGE and transferred to a nitrocellulose blotting membrane (PALL Corporation, Port Washington, USA). Membranes were blocked for 1 h in 1 × TBS containing 10% non-fat milk, 0.1% Tween 20 and incubated overnight with primary antibodies: rabbit anti-Ccnd2 (Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-Gapdh (KangChen Bio-tech Inc., Shanghai, China). Then membranes were incubated with IRDye® 680-conjugated goat anti- rabbit, IRDye 800CW-conjugated goat anti-mouse. Fluorophore-conjugated antibodies were detected using the Odyssey® Imager (LI-COR Biosciences Inc., Lincoln, USA).
8
Western Blot Analysis of Autophagy Markers
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Cells were collected and lysed with RIPA buffer (sc‐24948, Santa Cruz Biotechnology). Equal amounts of protein (40 µg) were separated on 10% or 12% SDS‐polyacrylamide gels and then transferred to a nitrocellulose blotting membrane (PALL Corporation, Mexico) or a PVDF blotting membrane (Bio‐Rad Laboratories, Hercules, CA, USA). After blocking with 5% non‐fat milk, the membrane was incubated overnight with the respective primary antibody at 4 °C. The following antibodies were obtained from Cell Signaling (Danvers, MA, USA): anti‐LC3B (1:1000, #83 506), anti‐ATG5 (1:1000, #12 994), anti‐ATG7 (1:1000, #2631), anti‐pAMPKα (1:1000, #2535), anti‐AMPKα (1:1000, #5832), anti‐cleaved caspase 3 (1:1000, #9661). The control anti‐β‐actin (1:10000, sc‐47778) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Signal was detected using the Bio‐Rad ChemiDoc system and signal density was measured using the Bio‐Rad Image Lab Software (Bio‐Rad Laboratories, Redmond, WA, USA).
9
Western Blot Analysis of Protein Expression
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Cells were collected and lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), protease inhibitors). Equal amounts of proteins (40 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis gels and then transferred to a nitrocellulose blotting membrane (PALL Corporation, Mexico). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 °C with the respective primary antibody. The following antibodies were used: anti-c-MYC (1:1000, 13987S), anti-SLC3A2 (1:1000, 13180S), anti-E-cadherin (1:1000, 3195S), and anti-SLC7A11 (1:500, 12691S) from Cell Signaling Technology (Danvers, MA); anti-β-actin (1:10,000, sc-47778), anti-MTDH (1:250, 517220), and anti-GPx4 (1:500, sc-166570) from Santa Cruz Biotechnology; anti-vimentin (1:1000,v6389) from Sigma-Aldrich; anti-ZEB1 (1:1000, NBP1-88845) from Novus Biologicals USA. Membranes were further incubated with secondary antibody (1:10,000, 7074S or 7076S, Cell Signaling Technology) at room temperature for 2 h. Signal bands were detected using the Bio-Rad ChemiDoc system and densitometry were analyzed with Bio-Rad Image Lab Software (Bio-Rad Laboratories).
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