Protein was extracted using Mammalian Protein Extraction Reagent (Thermo Fisher, Waltham, Mass) with protease and phosphatase inhibitors and electrophoresed on NuPage Novex 4–12% Bis-Tris Protein Gels, transferred to PVDF membranes, and blocked for one hour with 5% w/v BSA or 5% nonfat milk in 1X TBS and 0.1% Tween-20. Immunoblots were performed using rabbit anti-IQGAP1 (Abcam ab86064, Abcam, Cambridge, Mass), anti-pERK1/2 (Cell Signaling CST9101S, Cell Signaling, Danvers, Mass), and anti-ERK1/2 (Cell Signaling CST9102) polyclonal, and mouse anti-GAPDH monoclonal (Abcam ab8245) antibodies. Proteins were detected using the Amersham ECL reagent (GE Healthcare Life Sciences, Pittsburgh, Pa) on HyBlot CL film (Denville Scientific, Holliston, Mass). Densitometric analysis of protein expression was performed using ImageLab software (BioRad, Hercules, Calif).
Hyblot cl film
Manufactured by Thomas Scientific
Sourced in United States
The HyBlot CL film is a high-quality, chemiluminescent imaging film designed for detecting and capturing protein or nucleic acid signals in western blotting, northern blotting, and other molecular biology applications. It provides consistent and reliable results for visualization and documentation of blotting experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Non fat milk, by Bio-Rad (3 mentions)
Ab19857, by Abcam (2 mentions)
Micromethod of bradford, by Bio-Rad (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Anti rabbit or anti mouse secondary antibody, by Merck Group (2 mentions)
Nupage lithium dodecyl sulfate buffer, by Thermo Fisher Scientific (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Ab86064, by Abcam (1 mentions)
Bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Amersham ecl reagent, by GE Healthcare (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ab8245, by Abcam (1 mentions)
Chemiluminescent reagent, by GE Healthcare (1 mentions)
27 protocols using hyblot cl film
1
Western Blot Analysis of IQGAP1, pERK1/2, and ERK1/2
2
Western Blot Protein Extraction and Analysis
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After the treatments, the cells were harvested and washed twice with PBS. RIPA buffer containing a protease inhibitor and anti-phosphatase cocktail inhibitors II and III (Sigma Chemicals) was added for protein extraction. Proteins (10–25 μg) were mixed with equal amounts of 2× sample buffer (20% glycerol, 4% sodium dodecyl sulfate, 10% 2-βME, 0.05% bromophenol blue, and 1.25 M Tris–HCl, pH 6.8). The proteins were fractionated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane using a semi dry Transfer Cell (Bio-Rad), which was run at 18 V for 1 h. The membranes were blocked for 1 h in 5% nonfat dry milk in TBST (Tris-buffered saline with 0.1% Tween 20) and washed. The PVDF membranes were incubated with the primary antibody overnight at 4°C. The blots were washed three times with TBST and incubated for 2 h with the HRP-conjugated secondary anti-IgG antibody (1/5,000 in 5% nonfat dry milk in TBST) at room temperature. After this incubation, the membranes were washed three times in TBST and incubated for 5 min with a chemiluminescent reagent (GE Healthcare) to visualize immunoreactive bands. Images were captured on Hyblot CL film (Denville; Scientific, Inc) using JPI automatic X-ray film processor model JP-33. Optical density analysis of signals was carried out using Image J software.
3
Protein Extraction and Western Blot Analysis
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Cells were lysed with 50 mM Tris⋅HCl (pH 7.6) containing 1% NP40, 30 mM NaCl, 1 mM EDTA, 150 mM NaCl, and 1× proteinase inhibitor cocktail (cat. # 4693159001; Millipore Sigma) or RIPA buffer supplemented with EDTA-free protease inhibitor cocktail (11873580001; Sigma-Aldrich) and 1 mM PMSF. Protein concentrations were determined using Protein Assay Kit (5000002; BioRad). Lysates were resolved on 10% NUPAGE Bis-Tris gels (Invitrogen), transferred to polyvinylidene fluoride membranes, and incubated with the relevant antibodies. Stained membrane signals were visualized by using Hyglo HRP detection kit (cat. # E2500; Denville Scientific) and exposed to HyBlot CL film (cat. # E3012; Denville Scientific) by LICOR Odyssey Fc (LI-COR Biosciences). Protein levels were quantified using ImageJ (https://imagej.nih.gov/ij/ ). Details on antibodies used are listed in Table S8 .
4
Western Blot Analysis of Protein Lysates
5
Western Blot Protein Detection
6
Northern Blotting of Total RNA
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Total RNA (10 μg) was separated on a 8% polyacrylamide-7M urea gel in 1X TBE (90 mM Tris-borate 2 mM EDTA). The RNA was transferred to a Zeta-Probe GT blotting membrane (Bio-Rad) at 20V for ∼16 h at 4°C in 0.5X TBE. After transfer, membranes were allowed to dry, UV cross-linked on both sides, and incubated overnight at 45°C in UltraHyb (Ambion) hybridization buffer and oligonucleotides 5′-end-labeled with 32P-ATP with T4 polynucleotide kinase (New England Biolabs). Subsequently, membranes were washed once with 2X SSC (150 mM NaCl 15 mM sodium citrate) 0.1%SDS, incubated 10 min at 45°C in 2X SSC 0.1%SDS, and washed 5X with 0.2X SSC 0.1% SDS. After washing, air-dried membranes were exposed to HyBlot CL film (Denville Scientific) at −80°C.
7
Englerin A Modulates Stress Signaling Pathways
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A498 cells were plated in complete RPMI at 1.2 x 106 and 2.0 x 106 cells per T-75 flask for control and treated cells, respectively. After allowing cells to attach overnight, cells were treated with 0.1% DMSO or 100 nM englerin A for 3, 8, and 24 h. Cell extracts were prepared and Western Blot analysis was conducted as described previously [22 (link)]. Antibodies against MKK4, p-MKK4, eIF2α, p- eIF2α, p-JNK, and B-actin were obtained from Cell Signaling Technology (Danvers, MA) and that against JNK was obtained from Abcam (Cambridge, MA). Except for anti-JNK (diluted 1:2000), all antibodies were diluted 1:1000. Bands were visualized using SignalFire Plus ECL enhanced chemiluminescent substrate (Pierce) and exposed to HyBlot CL film (Denville Scientific). The film was developed with a Kodak film developer.
8
DDK Kinase Activity Inhibition Assay
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20 ng of purified human DDK was pre-incubated with increasing concentrations of each DDK inhibitor for 5 min. Then 10 µCi (γ)-32P ATP and 1.5 µM cold ATP were added in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT and incubated for 30 min at 30°C. The proteins were denatured in 1X Laemmli buffer at 100°C followed by SDS-PAGE and autoradiography on HyBlot CL film (Denville Scientific, Inc.). Auto-phosphorylation of DDK was used as an indicator of its kinase activity. 32P-labeled bands were quantified using ImageJ and the IC50 values were calculated using GraphPad (Prism 6).
9
Protein Extraction and Western Blot Analysis of Nuclear OCT4
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Protein extracts were prepared from purified nuclear fractions of MSCs. Briefly, cells were resuspended in lysis buffer containing 50 mM Tris–HCl (pH = 7.5), 1% Triton X-100, 137.5 mM NaCl, 10% Glycerol, 1 mM sodium vanadate, 1X protease inhibitors cocktail (Sigma). Lysates were incubated on ice for 15 min and centrifuged at 10,000 g for 15 min at 4 °C. Supernatant was collected (cytoplasmic fraction) and the nuclear pellet was separately resuspended in the same lysis buffer containing 0.2% SDS and briefly sonicated. Nuclear lysates were centrifuged at 10,000 g for 15 min at 4 °C and the supernatant was collected (nuclear fraction). Protein concentrations were determined using the micromethod of Bradford (Bio-Rad, CA). Nuclear samples (20 µg protein) were separated on a 10% SDS-PAGE gel and transferred to a HyBlot CL film (Denville Scientific, NJ, USA). Membranes were blocked using 1X PBST with 5% (w/v) non-fat dried milk for 1 h and hybridized overnight at 4 °C with an anti-OCT4 primary antibody (1:800, ab19857, Abcam). After washing with TBST buffer, membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody and washed five times for 5 min with TBST buffer. Bound antibodies were visualized using ECL Plus Western Blotting detection system (GE Healthcare), according to the manufacturer’s instructions.
10
Western Blot Protein Detection Methodology
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After the appropriate treatments, cells were washed with PBS and harvested in RIPA buffer (Millipore, MA, USA) containing protease inhibitor and anti-phosphatase cocktails, as previously described (14 (link)). Equal amounts of proteins were mixed with the same volume of 2× sample buffer, separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene di-fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) at 20 V for 60 min using a semi dry transfer cell (Bio-Rad) as previously described (14 (link)). The PVDF membrane was blocked with 5% dry milk in Tris-buffered saline with 0.1% Tween-20, rinsed, and incubated with primary antibody overnight. The blots were washed and incubated with HRP-conjugated secondary anti-IgG antibody. Membranes were washed and immunoreactive bands were visualized using a chemiluminescent substrate (ECL-Plus, GE Healthcare, Pittsburgh, PA, USA) (15 (link), 16 (link)). Images were captured on Hyblot CL film (Denville, Scientific Inc., Metuchen, NJ, USA). Optical density analysis of signals was performed using ImageQuant software (version 5, Molecular Dynamics).
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