Macrophages from pulpitis and healthy pulps which were treated with HMGB1 were lysed and then electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PVDF; PerkinElmer, USA). Membranes were blocked with 5% (w/v) non-fat dry milk, 1% (v/v) Tween 20 in PBS for 1 hr at room temperature and incubated overnight with commercially available Anti-Nf-KB antibody (1:1000) (Abcam, USA) at 4 °C. Detection was performed with electrochemiluminescence (ECL) and the blots were quantified by densitometry using an image analysis program (Amercontrol Biosciences, USA).
Polyvinylidene fluoride (pvdf)
Manufactured by PerkinElmer
Sourced in United States
PVDF is a type of lab equipment used for various scientific applications. It is a fluoropolymer material with a high resistance to chemicals, solvents, and extreme temperatures. PVDF is commonly used in the construction of laboratory components, such as tubing, membranes, and other equipment parts.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf kb antibody, by Abcam (2 mentions)
Bm chemiluminescence detection kit, by Roche (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (1 mentions)
α tubulin, by Merck Group (1 mentions)
Orf45, by Santa Cruz Biotechnology (1 mentions)
K bzip, by Santa Cruz Biotechnology (1 mentions)
Ucd 200, by Diagenode (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using polyvinylidene fluoride (pvdf)
1
NF-κB Activation in Pulpitis Macrophages
2
Quantification of Secreted Proteins in Co-Culture
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In co-culture system (BMSCs plus OBs), KG-1 cell lines were treated with CUR and/or CUR+ DNR for 24 h without FBS. After that media were collect and secreted proteins were deposited in this media by Trichloroacetic acid (TCA) method (Santa et al., 2016). Protein solution were resolved on 10% SDS-PAGE and then transferred electrophoretically to the polyvinylidene difluoride membrane (PVDF, Perkin-Elmer, Life Science, MA, USA) as described before (Mohammadi et al., 2016b). Membranes were subjected to immunodetection procedure using specific antibodies against β-Actin, OPN, CXCL-12 and 1L-6. Proteins were detected using a BM chemiluminescence detection kit (Roche Applied Science, Peuzberg, Germany).
3
Immunoprecipitation and Western Blotting
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Cells were harvested in lysis buffer (Cell Signaling) with protease inhibitors (10 µg/mL aprotinin/5 mM PMSF/10 µg/mL pepstatin/10 µg/mL leupeptin) as described previously [16 (link)]. The resulting lysates were subjected to IP and Western blot analysis. IP experiments were performed using anti-myc, anti-HA, or anti-AMPK antibodies and protein G Plus/protein A-agarose (GE, Pittsburgh, PA USA) as described previously [7 (link)]. GST pull-down was performed as described previously [3 (link)]. For Western blot analysis, proteins from each sample were separated on 4–12% NuPAGE Bis-Tris gels, and transferred onto polyvinylidene difluoride membranes (PVDF, Invitrogen) for 3 h. Then the gels were stained with Coomassie blue to assure transfer efficiency. The PVDF membranes were blocked with 5% non-fat milk and incubated with various primary antibodies as listed in Section 4.1 for 1 to 4 h at room temperature. The goat anti-mouse or goat anti-rabbit horseradish-peroxidase conjugated antibodies were used as secondary detection antibodies for 1 h of incubation. Enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) were incubated for 1 min to detect the proteins in PVDF membranes.
4
Western Blot Protein Detection Protocol
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Cells were collected in PBS and lysed in PBS containing 0.5% Nonidet P-40 (NP-40) containing 1X protease inhibitor cocktail (Roche, 4693132001). The cell lysates were sonicated 5 times (pulsed for 30 sec with 30 sec interval) using a Bioruptor (Diagenode, UCD-200) at low power setting. Total cell lysate (TCL) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes (PVDF; PerkinElmer, 1002001), blocked with 5% nonfat skim milk or 5% BSA (UniRegion, UR-BSA001-50G) in 1X TBST, and probed with primary antibody at 4 oC for 16 hours. After incubating the membrane with horseradish peroxidase (HRP) linked secondary antibody (GE healthcare, NA934 and NA931), the protein expression levels were detected by Pierce ECL Western Blotting Substrate (Thermo, 32106). Primary antibodies against KDM4A (Polyclonal antibody purified from rabbit) [12 (link)], K-bZIP (1:1000; Santa Cruz Biotechnology, sc-69797), Orf45 (1:1000; Santa Cruz Biotechnology, sc-53883), HA tag (1:4000; Cell Signaling Technology, #3724) and α-Tubulin (1:4000; Sigma, T6074-200UL) were used in this study.
5
Quercetin Modulates MAPK Signaling in Pulpitis
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T cells from pulpitis, treated with quercetin, were lysated and then electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PVDF; PerkinElmer, USA). Membranes were blocked with 5% (w/v) non-fat dry milk, 1% (v/v) Tween 20 in PBS for 1h at room temperature and incubated overnight with commercially available anti-MAPKs antibody (1:1000) (Abcam, USA) at 4oC. Detection was performed with electrochemiluminescence (ECL) and the blots were quantified by densitometry using the image analysis program (Amercontrol Biosciences, USA).
6
HMGB1-induced NF-κB activation in macrophages
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Macrophages from GCP and normal tissues, which were treated with HMGB1, were lysed and then underwent electrophoresis on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PVDF; PerkinElmer, USA). The membranes were blocked with 5% (w/v) non-fat dry milk, 1% (v/v) Tween 20 in PBS for 1 hour at room temperature and incubated overnight with commercially available Anti-Nf-KB antibody (1:1000) (Abcam, USA) at 4°C. Detection was performed with electrochemiluminescence (ECL) and the blots were quantified by densitometry, using an image analysis program (Amercontrol Biosciences, USA).
+ Expand
Macrophages from GCP and normal tissues, which were treated with HMGB1, were lysed and then underwent electrophoresis on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PVDF; PerkinElmer, USA). The membranes were blocked with 5% (w/v) non-fat dry milk, 1% (v/v) Tween 20 in PBS for 1 hour at room temperature and incubated overnight with commercially available Anti-Nf-KB antibody (1:1000) (Abcam, USA) at 4°C. Detection was performed with electrochemiluminescence (ECL) and the blots were quantified by densitometry, using an image analysis program (Amercontrol Biosciences, USA).
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