Cells were extracted in lysis buffer (0.5% Nonidet P-40, 5% sodium deoxycholate, 50 µM NaCl, 10 µM Tris-HCl, pH 7.5, 1% bovine serum albumin) and centrifuged at 4°C and 18,407 × g for 15 min. The supernatant was mixed with 2X loading buffer and boiled for 5 min, and then the samples were separated via 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% fat-free milk at room temperature for 1 h, incubated at 4°C overnight with antibodies S12 for LMP1 (1:50), anti-β-actin (1:10,000; cat. no. A1978; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), anti-G protein (1:500; cat. no. 371818; Sigma-Aldrich; Merck KGaA) and anti-heterogeneous nuclear ribonucleoprotein A/B (1:500; cat. no. sc-376411; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), washed, and then incubated with peroxidase-conjugated secondary antibody (1:2,000; cat. no. sc-11001; Santa Cruz Biotechnology, Inc.). Immune complexes were detected using an Amersham ECL Western Blotting Detection kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and gel imaging analysis (Cool Imager; Viagene Biotech, Inc). To confirm the expression levels of each protein examined in LMP1-transfected cells, western blotting for each protein was performed in triplicate.
Amersham ecl western blotting detection kit
Manufactured by Cytiva
Sourced in United Kingdom
The Amersham ECL Western Blotting Detection Kit is a laboratory equipment used for the visualization and detection of proteins in western blotting analysis. It utilizes a chemiluminescent substrate to generate a light signal that can be captured and quantified.
Automatically generated - may contain errors
Lab products found in correlation
Amersham ecl western blotting, by Cytiva (8 mentions)
Hybond, by Cytiva (4 mentions)
β actin, by Merck Group (2 mentions)
Ez link hpdp biotin, by Thermo Fisher Scientific (2 mentions)
Amersham hybond n positively charged nylon membrane, by Cytiva (2 mentions)
Spectrolinker xl 1500, by Spectroline (2 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Gapdh, by GeneTex (1 mentions)
Hybond p membrane, by Cytiva (1 mentions)
P8340, by Merck Group (1 mentions)
Ab105802, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Lamin a, by Merck Group (1 mentions)
8 protocols using amersham ecl western blotting detection kit
1
Western Blot Analysis of LMP1 Expression
2
Western Blot Analysis of SARS-CoV-2 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SARS-CoV-2-infected Vero E6 cells were lysed using the RIPA buffer supplemented with protease inhibitor (Merck Millipore, Billerica, MA, USA) and incubated at 100 °C for 5 min. A total of 15 μL of each sample was loaded into wells with the A YesBlot™ Western Marker I (10–200kDa) (WM1000 Smobio, Taiwan). Membranes were immunoreacted with IgYs against SARS-CoV-2 S protein subunits or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (GeneTex, Cat# GTX627408, 1:5000). Reactive bands were visualized by Amersham ECL Western Blotting Detection Kit (Amersham)
3
Antibody Validation for Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used in the western blot experiments: ANXA13, rabbit polyclonal (ab105802, Abcam, USA; the specificity of this antibody was further verify by a second ANXA13 antibody: goat polyclonal, AF4149-SP, R&D System, USA); β-actin, mouse monoclonal (clone AC-74, A2228, Sigma); MMP9, rabbit polyclonal (ab38898, Abcam); Phospho-Akt (Thr308) (244F9), Rabbit mAb #4056 (Cell Signaling Technology). Protein lysates were prepared from cultured cells in ice-cold Tris buffer (20 mmol/L; pH 7.5) containing NaCl (137 mmol/L), EDTA (2 mmol/L), Triton X (1%), glycerol (10%), NaF (50 mmol/L), DTT (1 mmol/L), and a protease inhibitor cocktail (P8340, Sigma, USA). After electrophoresis (SDS-PAGE), protein samples were transferred onto Hybond-P membranes (Amersham Biosciences). The blot was blocked in TBS with 5% nonfat milk and 0.1% Tween 20, followed by incubation with primary antibody (RT 1h) and HRP-tagged goat anti-rabbit or -mouse IgG secondary antibody (sc-2030 or sc-2357, Santa Cruz, USA). The blots were visualized and quantitated using Amersham ECL Western Blotting Detection Kit (Amersham Biosciences).
4
SARS-CoV-2 Spike and Nucleocapsid Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein lysates of SARS-CoV-2-infected cells were dissolved with RIPA buffer. The protein expression was analyzed by Western blot analysis in which the TGN Gel 4–15% (QP5510, SMOBIO) with an SE260 Mighty Small II Deluxe Mini Vertical Protein Electrophoresis Unit (Hoefer, Inc., Holliston, MA, USA) was used. A YesBlot™ Western Marker I (10–200 kDa) (WM1000 Smobio, Taiwan) was used as a molecular weight marker. Antibodies including the mouse monoclonal anti-spike (1:5000; GeneTex Cat #GTX632604) and mouse monoclonal anti-nucleocapsid (1:2000; GeneTex Cat# GTX632269) antibodies were used to detect the SARS-CoV-2 spike and nucleocapsid proteins, respectively. The mouse monoclonal GAPDH antibody (GTX627408, GeneTex, 1:5000) was used as the loading control. The anti-chicken IgY (IgG) (whole molecule)–peroxidase antibody produced in rabbits (Sigma-Aldrich A9046) and the anti-rabbit IgG (whole molecule)–peroxidase antibody produced in goats (Sigma-Aldrich A0545) were used as the secondary antibodies as required. The luminol-based detection of Western blot signals was performed using the Amersham ECL Western Blotting Detection Kit (Amersham Biosciences, Buckinghamshire, UK). The images were captured and analyzed using Image Quant™ LAS 4000 (GE Healthcare, Chicago, IL, USA).
5
Western Blot Analysis of Pbp2c Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from crude extracts (10 μg) were separated on 15% SDS-PAGE, transferred to a nitrocellulose membrane (Hybond, Amersham Biosciences, Little Chalfont, United Kingdom), and incubated with mouse anti-Pbp2c antiserum (1/5,000). Nitrocellulose membranes were incubated in 10 mM Tris-Cl buffer pH 7.5 containing 150 mM NaCl, 0.025% Tween 20, and 2.5% nonfat dry milk for 1 h at 4°C. Peroxidase-conjugated goat anti-mouse antibodies (Life Technologies, Saint-Aubin, France; 1/6,000) were used as secondary antibodies. Pbp2c-antibodies complexes were detected by chemiluminescence (Amersham ECL Western Blotting Detection Kit, Cytiva) using a film exposure time of 5 min. Purified His-tagged-Pbp2c (3 ng) was used as the positive control.
6
RNA Labeling and Detection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with EZ-Link HPDP-Biotin (Thermo Scientific), 500 ng of heat denatured (for 10 minutes at 65°C) total RNA were spotted on an Amersham Hybond-N+ positively charged nylon membrane (Cytiva). The RNA was ultraviolet-cross-linked to the membrane with Spectrolinker XL-1500 (Spectroline, ‘optimal crosslink’) and blocked for 10 minutes in blocking solution (PBS pH 7.5, 10% SDS, 1 mM EDTA). The membrane was then incubated in 1:1000 dilution of streptavidin-HRP (Pierce) in blocking buffer for 15 minutes. After washings with decreased concentration of SDS, the signal was recorded (Amersham ECL Western Blotting Detection Kit, Cytiva).
7
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell protein was prepared using RIPA Lysis Buffer (Beyotime Institute of Biotechnology). Protein was measured using a BCA protein Assay Kit (CWBIO). Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes using a Bio-Rad Mini PROTEAN 3 system (Bio-Rad Laboratories, Inc.). The membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 at room temperature for 1 h. The primary antibodies were as follows: β-actin (mouse monoclonal, dilution 1:10,000; A4551) was from Sigma-Aldrich; Merck KGaA. Lamin A (mouse monoclonal, dilution 1:1,000; sc-71481) was obtained from Santa Cruz Biotechnology, Inc. Anti-NF-κB p65 (rabbit polyclonal, dilution 1:1,000; ab16502), anti-Histone H3 acetyl K9 (rabbit polyclonal, dilution 1:5,000, ab4441), anti-HDAC1(rabbit polyclonal, dilution 1:5,000, ab109411) and anti-IκB-α (rabbit polyclonal, dilution 1:5,000, ab32518) were purchased from Abcam. Horseradish peroxidase-conjugated anti-mouse (1:2,500 dilution) or anti-rabbit (1:2,500 dilution) secondary antibodies were purchased from Bioworld Technology, Inc. Immunoreactive bands were visualized by using the Amersham ECL Western Blotting Detection Kit (Cytiva) according to the manufacturer's instructions. β-actin served as a loading control (19 (link),23 (link)).
8
RNA Labeling and Detection Assay
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!