Five hundred mL cultures of C. bescii strains (JWCB017, 049, and 054) were grown to mid-log phase at 75 °C, harvested by centrifugation at 6000×g at 4 °C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4× freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (75 µg) were electrophoresed in 4–15 % gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.
Ecl western blotting substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL Western Blotting Substrate kit is a reagent used in the process of Western blotting to detect and quantify specific proteins in a sample. The kit contains the necessary components to generate a chemiluminescent signal that can be detected and measured.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
His tag 6xhis monoclonal antibody, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Cell lytic b cell lysis reagent, by Merck Group (2 mentions)
Mini protean 3, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Imagequant las 4000 camera system, by GE Healthcare (1 mentions)
Ab6721, by Merck Group (1 mentions)
Hsp90α, by Abcam (1 mentions)
Las 4000, by Cytiva (1 mentions)
Ab32360, by Abcam (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab2068, by Abcam (1 mentions)
Pvdf membrane, by Carl Roth (1 mentions)
28 protocols using ecl western blotting substrate kit
1
Proteomic Analysis of Thermophilic C. bescii
2
Protein Extraction and Analysis from C. bescii
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five hundred mL cultures of C. bescii were grown to mid-log phase at 65, 70 or 75°C, harvested by centrifugation at 6,000×g at 4°C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4X freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (80 µg) were electrophoresed in 4–15% gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5,000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.
3
Western Blot Analysis of Stress Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of protein samples were separated using the SDS-PAGE technique and afterward transferred onto nitrocellulose membranes. Ponceau S staining was used for electro-transfer uniformity and normalization purposes. Thereafter, the membranes were washed and blocked with 2% BSA in TRIS-buffered saline containing 0.05% Tween 20, pH 7.6, and exposed for 2 h to the primary calreticulin (Thermo Scientific, catalog # PA3-900), HSP 90-α (Abcam, Cambridge, UK, catalog # ab13492), HSP 60 (Thermo Scientific, catalog # MA3-013), HSPB1 (Abcam, catalog # ab79868), and Annexin A1 (Thermo Scientific, catalog # PA5-22266) antibodies in TBS with 1% BSA followed by the appropriate IgG coupled with horse radish peroxidase (IgG–HRP) secondary antibodies for 1 h (Abcam, catalog # ab6721, and Sigma-Aldrich, catalog # A2304). The subsequent chemiluminescence reaction was revealed using the ECL Western Blotting Substrate kit (Thermo Scientific) and images were taken with the Image Quant LAS 4000 camera system (GE Healthcare, Uppsala, Sweden). Thereafter, Digital densitometry analysis was performed using the ImageJ analysis freeware. All original, unaltered, and unprocessed membrane and Ponceau S-colored membrane images can be found in Figures S1–S6 as Supplementary Material .
4
Hippocampal TLR4-Myd88-NF-κB Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hippocampal tissue was grid and centrifuged in a pre-chilled tissue lysate at 12,000 rpm for 30 min. The supernatant of total protein was extracted for SDS-PAGE and the protein was semi-dried for membrane transfer. The cells were blocked 2 h and incubated overnight at 4°C with TLR4 (ab22048, Abcam, Cambridge, MA, USA), Myd88 (ab2068, Abcam, USA), NF-κB antibody (ab32360, Abcam, USA), followed three washes and incubation with secondary antibody for 1 h. After four washes using TBST, cells were developed with ECL Western Blotting Substrate kit (32109, Pierce™, Thermo Fisher Scientific, Waltham, MA, USA) and gray value was measured by using Quantity One software.
5
Quantifying Protein Abundance by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated from 200 mg of seedlings with Laemmli Buffer. Total proteins weighing 15 µg were separated by 12% SDS-PAGE and transferred to polyvinyl difluoride (PVDF) membranes (Roth, Karlsruhe, Germany) by wet blotting at 4°C in 25 mM Tris-HCl at pH 8.3, 24 mM glycine and 10% ethanol for 2 h at 250 mA. Enhanced chemiluminescence detection was performed using an ECL Western Blotting Substrate kit as recommended by the manufacturer (Thermo Scientific, Rockford, USA). Mouse anti-GFP antibodies [632381(JL-8), Clontech], at a dilution of 1:1000, were used for immunoblotting. Secondary antibodies were Alexa Fluor® 555 goat anti-mouse (Fig. 5A ) SFX Kit (A31621, Invitrogen Life Technologies) and goat anti-mouse HRP (Fig. 5B ) (170–6516, Biorad, Solna, Sweden). Western blots were performed independently three times and gave consistent results; one representative blot is shown.
6
Western Blot Analysis of AMPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedure of western blot analysis was performed as described previously
[22] (link). Firstly, total proteins were extracted using RIPA lysis buffer (Thermo Scientific) containing proteinase inhibitors, phosphatase inhibitors, and PMSF. Secondly, protein concentration was measured using BCA Protein Assay kit (Thermo Scientific), and the protein samples were prepared using 6×SDS protein loading sample buffer. Thirdly, equal amounts of proteins (20‒40 μg/well) were loaded onto 10% SDS-PAGE gel and separated by electrophoresis, and then transferred onto PVDF membranes (Millipore, Billerica, USA). Subsequently, the membranes were blocked with 5% skimmed milk and incubated with primary antibodies, including anti-β-actin, total AMPKα, AMPKβ1, and AMPKβ2 antibodies, at 4°C overnight, followed by incubation with indicated HRP-conjugated secondary antibodies for 2 h at room temperature. Finally, protein bands were detected by using ECL western blotting substrate kit (Thermo Scientific).
[22] (link). Firstly, total proteins were extracted using RIPA lysis buffer (Thermo Scientific) containing proteinase inhibitors, phosphatase inhibitors, and PMSF. Secondly, protein concentration was measured using BCA Protein Assay kit (Thermo Scientific), and the protein samples were prepared using 6×SDS protein loading sample buffer. Thirdly, equal amounts of proteins (20‒40 μg/well) were loaded onto 10% SDS-PAGE gel and separated by electrophoresis, and then transferred onto PVDF membranes (Millipore, Billerica, USA). Subsequently, the membranes were blocked with 5% skimmed milk and incubated with primary antibodies, including anti-β-actin, total AMPKα, AMPKβ1, and AMPKβ2 antibodies, at 4°C overnight, followed by incubation with indicated HRP-conjugated secondary antibodies for 2 h at room temperature. Finally, protein bands were detected by using ECL western blotting substrate kit (Thermo Scientific).
7
Western Blot Analysis of Autophagy Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with different concentrations of EHHM in the presence or absence of 3-MA (5 mM) or Baf A1 (10 nM) for 24 h, and then lysed in lysis buffer [50 mM Tris-HCl (pH 6.8), 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 10 mM dithiothreitol, 1 mM phenylmethane sulfonyl fluoride and 1X protease inhibitor cocktail (Roche, Basel, Switzerland)]. Equal amounts of protein samples were quantified using the RC DC ™ Protein Assay Kit I (Bio-Rad Laboratories, Hercules, CA, USA) and electrophoresed by 8–12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked with a 5% non-fat milk powder solution in Tris-buffered saline with Tween 20 (TBST) buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.05% Tween 20] for 1 h at room temperature and then incubated with the corresponding primary antibodies (1:1,000) overnight at 4°C. Upon washing with TBST, membranes were incubated with horseradish peroxidase-conjugated anti-mouse (cat. no. 31430; 1:5,000) and anti-rabbit (cat. no. 31460; 1:5,000) IgG secondary antibodies (Thermo Fisher Scientific, Inc.) for 2 h at room temperature and detected with ECL Western Blotting Substrate kit (Thermo Fisher Scientific, Inc.) (34 (link)).
8
Western Blotting of CSF Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as in our previous studies17 (link),18 (link). CSF cells were lysed with RIPA buffer (50 mM Tris-base, 10 mM EDTA, 150 nM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein lysates of the supernatant were quantified using the BCA protein assay kit (Thermo Scientific). Equal amounts of protein were separated by 10 and 15% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). After blocking the PVDF membranes with a double blocker (phosphate-buffered saline buffer-based BDP) for 1 h, the transferred PVDF membranes were incubated with the primary antibodies overnight at 4 °C. After washing in Tris-buffered saline containing 0.1% Tween-20 (TBS-T buffer), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and developed using an enhanced chemiluminescence (ECL) Western blotting substrate kit (Thermo Fisher Scientific). The antibodies used in this study are presented in Supplementary Table S3 .
9
Clostridium thermocellum Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
Clostridium thermocellum strains (JWCT02, JWCT06, JWCT07, and JWCT08) were grown to mid-log or stationary phase at 60 °C in 20 mL CTFUD-NY medium without uracil. Cells were harvested by centrifugation at 6000×g at 4 °C for 15 min, and cell pellets were washed using 50 mM Tris–Cl buffer (pH 8.0) and resuspended in Tris–Cl buffer to OD600 20. Cells were lysed by boiling in the presence of SDS [30 (link)]. Cell free extracts were electrophoresed in 4–15% gradient Mini-Protean TGX gels, that were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus and then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.
+ Expand
Clostridium thermocellum strains (JWCT02, JWCT06, JWCT07, and JWCT08) were grown to mid-log or stationary phase at 60 °C in 20 mL CTFUD-NY medium without uracil. Cells were harvested by centrifugation at 6000×g at 4 °C for 15 min, and cell pellets were washed using 50 mM Tris–Cl buffer (pH 8.0) and resuspended in Tris–Cl buffer to OD600 20. Cells were lysed by boiling in the presence of SDS [30 (link)]. Cell free extracts were electrophoresed in 4–15% gradient Mini-Protean TGX gels, that were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus and then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.
10
Protein Expression Analysis by Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were created using 100 μL of RIPA buffer unless otherwise stated. Proteins (50 μg) were resolved via gel electrophoresis on reducing SDS/PAGE run at 100V for 1 h. Transfer to nitrocellulose membranes was done at 100V for 1 h as well. The membranes were blocked in 5% skim milk dissolved in TBS‐T wash buffer (Tris‐buffered saline containing 0.1% Tween) at room temperature for 1 h. Following this, blots were incubated overnight at 4 °C with protein‐specific primary antibodies containing 5% BSA to β‐actin at 1 : 5000 (Sigma, Cat# A5441), KIAA0101 at 1 : 1000 (Abnova, Burlington, ON, Canada, Cat# H00009768‐M01), and AR Primary Ab (Santa Cruz, Mississauga, ON, Canada, Cat# sc‐7305) 1 : 500 in TBS‐T + 5% BSA. After incubation, blots were washed four times in TBS‐T for 5 min each. Lastly, blots were incubated with HRP‐conjugated anti‐mouse secondary antibody diluted in 10 mL of TBS‐T at room temperature for 1 h (β‐actin 1 : 5000, KIAA0101 1 : 1000, and AR 1 : 2000). After washing as above, chemiluminescence was determined in a GelDoc system using an ECL western blotting substrate kit (ThermoFisher, Burlington, ON, Canada Cat# 32016).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!