Cells were washed twice with PBS. To obtain whole cell extracts for immunoblotting, cells were resuspended with RIPA buffer (50 mM Tris-HCl, 250 mM NaCl, 1% TritonX-100, 0.25% Sodium deoxycholate, 0.05% SDS, 1 mM DTT) and lysed on ice for 20 min. For immunoprecipitation experiments, cells were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 10% glycerin) and incubated on ice for 20 min, then sonicated for 30 sec. For each sample, 250 μg total protein was incubated with anti-Flag agarose for 1.5 h at 4°C, then washed five times with lysis buffer. All samples were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Signals were detected with specific antibodies using eECL Western Blot Kit (CWBIO).
Eecl western blot kit
Manufactured by CWBIO
Sourced in China
The EECL Western Blot Kit is a laboratory equipment product designed for the detection and analysis of proteins in biological samples using the Western blot technique. The kit provides the necessary components and reagents required to perform this analytical procedure.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Bca protein assay kit, by CWBIO (4 mentions)
Il 1β, by Abcam (4 mentions)
Ripa buffer, by Solarbio (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Fusion fx6, by Vilber (2 mentions)
Immune blot pvdf membranes, by Roche (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by CWBIO (2 mentions)
Cw0264, by CWBIO (2 mentions)
Anti gfp, by Abmart (1 mentions)
Anti his tag mouse monoclonal antibody, by CWBIO (1 mentions)
Goat anti rat igg h l hrp, by Abcam (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Mmp 9, by Abcam (1 mentions)
Goat anti rabbit or mouse igg, by ZSGB-BIO (1 mentions)
Bca method, by Sangon (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Anti β actin, by Merck Group (1 mentions)
41 protocols using eecl western blot kit
1
Whole Cell Protein Extraction and Immunoprecipitation
2
BTG2 Protein Expression Analysis
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Cellular proteins were extracted from H460 cells using RIPA buffer (Solarbio), separated on a 15% SDS PAGE gel and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% nonfat dry milk in TBST for 1 h at room temperature. Next, the membranes were incubated with anti‐BTG2 (diluted 1: 100, Santa Cruz) and anti‐GAPDH (diluted 1:5000, Proteintech) overnight at 4°C. The membrane was then incubated with Rabbit IgG HRP‐conjugated Antibody (diluted 1:5000, Proteintech) diluted in TBST at 37°C for 40 min. Protein signals were detected using the eECL Western Blot Kit (CWBio).
3
Protein Expression and Western Blot Analysis
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HEK293F cells were transfected with same amounts of plasmids of C-terminal GFP tagged NALCN (WT/mutant)–FAM155A–UNC79–UNC80 in the same ratio for protein expression. Cells were lysed 48 h post transfection in RIPA buffer (Solarbio) with 1.3 μg/mL aprotinin, 0.7 μg/mL pepstatin, 5 μg/mL leupeptin, and 2 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication. Lysates normalized by the expression level of GAPDH were loaded onto 14% sodium dodecyl sulfate–polyacrylamide gels for electrophoresis (SDS–PAGE). The separated proteins were then transferred to poly-vinylidene fluoride (PVDF) membranes (Merck Millipore). After blocking with 3% BSA for 1 h at 37 °C in Tris-buffered saline with Tween-20 (TBS-T), the membranes were incubated with the primary antibodies for 1 h at 37 °C and then with the horseradish peroxidase (HRP) labeled secondary antibody (1:5000 dilution, CWBIO) for 1 h. WB bands were visualized with the eECL western blot kit (CWBIO). The primary antibodies used were anti-GFP (1:3000 dilution, Abmart) and anti-GAPDH (1:5000 dilution, Proteintech).
4
Protein-Protein Interaction Assay
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The putative interactions between MdHDA19 and MdERF4C or MdERF4G were investigated using a pull-down assay. The respective recombinant proteins were fractionated on a sodium dodecyl sulphate (SDS)–polyacrylamide gel using a Tricine–SDS–polyacrylamide gel electrophoresis Gel Kit (Cwbio, code no. CW2384) and polyvinylidene fluoride membranes (Millipore, code no. HATF00010). The membranes were then incubated with either an anti-His-tag mouse monoclonal antibody (Cwbio, code no. CW0286M), diluted 1:3,000 in TBST (Tris, Buffer, Solution, Tween) buffer (10-mM Tris-base, pH 8.0, 150-mM NaCl, 3 g/100-mL bovine serum albumin, 50-µL/100 mL Tween-20), or with an anti-GST (Abcam, code no. ab92) antibody diluted 1:5,000 in TBST. The secondary antibody was a Goat Anti-Rat IgG H&L (HRP; Abcam, code no. ab205719), which was diluted 1:10,000 in TBST buffer. The eECL Western Blot Kit (Cwbio, code no. CW0049S) was used for visualization using a chemiluminescence apparatus (Amershan Imager 680, GE, USA).
5
MucR1 Homodimer Formation Analysis
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To test the ability of MucR1 to form homodimer in SF2 derivatives carrying sacB in pA, rhizobial cells (SF2MucR1FLAG, MucR1-FLAG: L-GC, MucR1-FLAG: M-GC, and MucR1-FLAG: H-GC) were cultured in 50 mL TY medium to an OD600 of 1.2. Formaldehyde was added at a final concentration of 1% in the culture which was then shaken at 28 °C, 100 rpm for 15 min to allow crosslinking. The crosslinking reaction was terminated by adding a final concentration of 100 mM glycine (28 °C, 100 rpm, 5 min). 1 mL of the above solution was centrifuged (5000 × g, 4 °C, 1 min), resuspended in 50 µL SDS loading buffer to a uniform cell density, and then boiled for 10 minutes for lysis. Next, lysates were separated on 12% SDS-PAGE and transferred to a nitrocellulose membrane. For immunodetection of individual proteins, the method described previously was used [30 (link)]. Briefly, mouse monoclonal Anti-FLAG M2 antibody (Sigma), HRP (horseradish peroxidase) conjugated goat Anti-mouse IgG (Abcam), and eECL Western blot kit (CWBIO, Beijing, China) were used, and chemiluminescence signals were visualized using Fusion FX6 (Vilber) and Evolution-Capt Edge software.
6
Western Blot Analysis of Proteins
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The protein extract was dissolved in a cell lysis buffer containing 1% protease inhibitor. Protein concentration of the homogenized lysates was measured using a BCA method (Sangon, Shanghai, China). Equal amount of protein was separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were probed overnight at 4°C with the following primary antibodies: rabbit polyclonal antibody against human TIPE2 (1:300; BOSTER, Wuhan, China), rabbit monoclonal antibody against VEGF (1:2000; Millipore, CA, USA), the matrix metalloproteinase 9 (MMP-9) and urokinase-PA (u-PA) (1:1000; EPITOMICS, Hangzhou, China), mouse monoclonal antibody against Rac1 (1:300; Abcam, Hongkong), β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat anti rabbit or mouse IgG, ZSGB-Bio) conjugated with peroxidase for 1 h at room temperature. After washing, signals were visualized by eECL Western Blot Kit (CWBIO).
7
Protein Expression Analysis by Western Blot
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Total cellular proteins were extracted, subjected to SDS-page electrophoresis and transferred to PVDF membranes using standard procedures. The primary antibodies included rabbit polyclonal anti-MMP-2 (1:1000; Abcam(Hong Kong) Ltd, HK, China) and mouse monoclonal anti-β-actin (1:1000; Sigma, St. Louis, USA); β-actin was used as an internal loading control. The bands were visualized and imaged using eECL Western Blot Kit (Cwbiotech, Beijing, China) and densitometry was performed using Image J (Version 1.49e, NIH, USA).
8
Monitoring MucR1 Protein Dynamics
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To monitor the protein levels of MucR1 and its derivatives at different growth phases, rhizobial cells cultured in TY medium were sampled every 3 h in a time-course manner that started from OD600 ≈ 0.3. The cell pellets were resuspended in corresponding volume of SDS-loading buffer to normalize the cell density and then lysed by boiling for 10 min. The lysates were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes. For immunodetection of individual proteins, mouse monoclonal anti-FLAG M2 antibody (Sigma), HRP (horseradish peroxidase)-conjugated Goat Anti-Mouse IgG (Abcam), and eECL Western Blot Kit (CWBIO) were used and chemiluminescence signals were captured and quantified using a Fusion FX6 (Vilber) and the Evolution-Capt Edge software.
9
Co-Immunoprecipitation and Western Blotting
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Total proteins were extracted using GTEN buffer as described by Du et al. (2021 ). For Co‐IP assays, for each 1.6 ml of total protein extraction a 12 µl of GFP‐trap_A beads (Chromotek) were added, and the mixtures were incubated for 2 hr with gentle shaking at 4 °C before proteins were spun down. The beads were washed with protein extraction buffer five times before being boiled in protein loading buffer. For western blot assays, proteins were separated on 10% sodium dodecyl sulphate (SDS)‐polyacrylamide gels and subsequently transferred to Immune‐Blot PVDF membranes (Roche). The membranes were blocked in 10 ml of blocking buffer (TBST; Tris‐buffered saline pH 7.2, 0.05% Tween 20 [Sigma]) containing 5% of nonfat milk, with gentle shaking for 2 hr at room temperature. Subsequently, membranes were incubated with anti‐GFP (#AE012, ABclonal), anti‐myc (#AE010, ABclonal), or anti‐pERK antibody at the appropriate dilution for 1.5–2 hr with gentle shaking at room temperature. Then the membranes were washed five times and incubated with the secondary antibodies horseradish peroxidase‐conjugated goat anti‐rabbit IgG (H + L) antibody (#AS014, ABclonal) for another 1.5–2 hr. After this incubation, membranes were washed with TBST five times before proteins were detected (eECL western blot kit; CWBio).
10
Western Blot Analysis of AKT/FoxO1 Signaling
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Total protein was extracted and quantified using the BCA method. About 40 μg of protein was loaded and separated with SDS-PAGE. PVDF membranes (Milipore) were blocked and incubated with primary antibodies against AKT, p-AKT (Phospho-Akt-Ser473), FoxO1, p-FoxO1 (Phospho-FOXO1-Ser256), Bim, and β-actin, all at a dilution of 1:1000. Finally, membranes were immunoblotted with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1:5000). All antibody reagents were purchased from Cell Signal Technology. Bands were detected with eECL Western Blot Kit (Cwbio, Beijing, China) and quantified using Image J software (Bio-Rad).
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