Deparaffinization and protein extraction of FFPE tissue was carried out using Qproteome FFPE Tissue Kit (Qiagen, Hilden, Germany; #1042481) following manufacturer’s instructions. Extracted proteins were determined by Bradford assay (BIO-RAD, CA, USA; #5000205). Quantified proteins were mixed with 5 × SDS-PAGE loading buffer (Biosesang, Seongnam, Korea; S2002) in concentration of 2 μg/μL and boiled at 95 °C for 5 min. Equal quantities of protein were separated to SDS-PAGE gel and transferred to nitrocellulose membranes (BIO-RAD; #1704158). Membranes were blocked by incubation in 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 and probed with antibody against ATX (1:1000; Abcam, Cambridge, UK; ab140915), LPA1(1:2000; Abcam; ab166903), and LPA2(1:1000; Abcam; ab38322) diluted in 1% BSA in TBS, 0.1% Tween 20, and 0.02% NaN3. The membranes were washed and then incubated with secondary antibodies (HRP conjugated anti-mouse IgG, or anti-rabbit IgG) (1:20,000; Santa Cruz, TX, USA) for 1 h at room temperature. The bands were visualized using WesternBright ECL (Advansata, CA, USA; K-12045-D50) after washing the membrane and exposed to X-ray film.
Sds page loading buffer
Manufactured by Biosesang
5X SDS-PAGE loading buffer is a concentrated solution used to prepare protein samples for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It contains the anionic detergent SDS, which denatures proteins and gives them a uniform negative charge. The buffer also includes reducing agents, such as dithiothreitol (DTT) or beta-mercaptoethanol, to break down disulfide bonds in proteins, and tracking dyes to monitor the progress of the electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Biosesang (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab140915, by Abcam (1 mentions)
Ab166903, by Abcam (1 mentions)
Ab38322, by Abcam (1 mentions)
Qproteome ffpe tissue kit, by Qiagen (1 mentions)
Hrp conjugated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Protein a agarose, by Santa Cruz Biotechnology (1 mentions)
Miracle star, by iNtRON Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Azure c600, by Azure Biosystems (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ho 1 antibody, by Abcam (1 mentions)
Lps l2630, by Merck Group (1 mentions)
P nf κb, by Santa Cruz Biotechnology (1 mentions)
7 protocols using sds page loading buffer
1
Extraction and Immunoblotting of Proteins from FFPE Tissue
2
Shp2 Protein Immunoprecipitation from Cell Lysates
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Cell lysate was extracted using RIPA buffer (Biosesang), (1 μM protease inhibitor, 10 μM sodium orthovanadate). After 1 h incubation on ice followed by centrifugation, whole cell lysate was acquired. 1 μg of Shp2 antibody (#VL3159027A, Invitrogen) was added in protein lysate and incubated at 4 °C for overnight. Protein A agarose (#sc-2001, Santa-cruz) was added in protein lysate and incubated at 4 °C for 4 h. Beads were gathered by centrifugation and protein sample was prepared by adding 5 × SDS-PAGE loading buffer (#SF2088-110-00, Biosesang), followed by boiling at 100 °C for 10 min.
3
Whole Cell Protein Extraction and Analysis
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Whole Cell lysate was extracted using RIPA buffer (Biosesang), supplemented with 1 μM protease inhibitor, 10 μM sodium orthovanadate. After 1 h incubation on ice followed by centrifugation, whole cell lysate was acquired. Quantification of proteins were performed with pierce BCA protein assay Kit (#23225, Thermo Fischer Scientific). Protein sample was prepared by adding 5 × SDS-PAGE loading buffer (#SF2088-110-00, Biosesang) and boiling at 100 °C for 10 min. Approximately 15 μg of each total protein was loaded and separated on 10% SDS-PAGE gel. Separated proteins were transferred to activated PVDF membrane. Membrane with transferred proteins was blocked with 5% skim milk in TBS-T in RT for 1 h followed by washing. Primary antibody (1:500 ~ 1:1000) in TBS-T was incubated with 1% sodium azide at 4 °C for overnight. After washing, the membrane was incubated with secondary antibody (1:10000) in TBS-T at RT for 1 h. Chemoluminescence was detected by Chemi-Doc using Miracle-Star (#16028, iNtRON Biotechnology) kit or West-Queen (#16026, iNtRON Biotechnology) kit.
4
Comparative Analysis of EV Isolation Methods
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EVs were isolated
from 50 μL of plasma by UC, Exoquick, and ExoPRISM methods.
Isolated EVs were extracted using RIPA buffer (Millipore, USA) with
protease inhibitor (Thermofisher, USA), and total protein concentrations
of EVs were determined by the BCA assay kit (Thermo Fisher, USA).
Equal volumes and equal amounts of total proteins were used for Western
blot analysis. EVs were boiled at 95 °C for 10 min with SDS-PAGE
loading buffer (Biosesang, Korea). All samples were separated on 8%
Tris-glycine SDS-PAGE gel and they were transferred to polyvinylidene
fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with
5% skim milk at room temperature for 1 h, and they were incubated
with primary antibodies against CD9, CD81, Albumin, and TSG101 (Table S3 ) at 4 °C overnight. The next day,
membranes were incubated with the corresponding secondary horse radish
peroxidase (HRP)-conjugated antibody at room temperature for 1 h.
Chemiluminescent detection of proteins was performed using Pierce
ECL Western Blotting Substrate (Thermofisher, USA), and blot imaging
was performed using Azure C600 equipment (Azure C600).
from 50 μL of plasma by UC, Exoquick, and ExoPRISM methods.
Isolated EVs were extracted using RIPA buffer (Millipore, USA) with
protease inhibitor (Thermofisher, USA), and total protein concentrations
of EVs were determined by the BCA assay kit (Thermo Fisher, USA).
Equal volumes and equal amounts of total proteins were used for Western
blot analysis. EVs were boiled at 95 °C for 10 min with SDS-PAGE
loading buffer (Biosesang, Korea). All samples were separated on 8%
Tris-glycine SDS-PAGE gel and they were transferred to polyvinylidene
fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with
5% skim milk at room temperature for 1 h, and they were incubated
with primary antibodies against CD9, CD81, Albumin, and TSG101 (
membranes were incubated with the corresponding secondary horse radish
peroxidase (HRP)-conjugated antibody at room temperature for 1 h.
Chemiluminescent detection of proteins was performed using Pierce
ECL Western Blotting Substrate (Thermofisher, USA), and blot imaging
was performed using Azure C600 equipment (Azure C600).
5
Anti-inflammatory Activity of Plant Extracts
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Chrysanthemum zawadskii, Peppermint (Mentha piperita) and Glycyrrhiza glabra were purchased from Kyeondong market. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin antibiotics were purchased from Gibco; Thermo Fisher Scientific, Inc. Griess reagent and LPS (L2630) were procured Sigma-Aldrich; Merck KGaA. Quanti-Max™ WST-8 Cell Viability Assay Kit has gotten from Biomax. Goat anti-mouse IgG (H+L) Alexa Fluor plus 488 conjugated secondary antibodies were purchased from Invitrogen; Thermo Fisher Scientific, Inc. Enzyme-linked immunosorbent assay (ELISA) kit PGE2, TNF-α, IL-6, and IL-1β were procured from R&D System. Mouse IFN Beta ELISA Kit (TCM, Serum) was supplied by BL Assay Science. HO-1 activity kit was purchased from Cusabio. Radio-immunoprecipitation assay buffer (RIPA buffer) came from Thermo Fisher Scientific, Inc. Bradford's assay reagent was purchased from Bio-Rad Laboratories, Inc. 5X SDS-PAGE loading buffer was purchased from Biosesang. Antibodies against iNOS, COX-2, p-NF-κB, p-IκB, p-Akt, p-STAT1, STAT1, and β-actin were purchased from Santa Cruz Biotechnology, Inc. HO-1 antibody was procured from Abcam. Horseradish peroxidase (HRP)-IgG secondary antibodies and diamidino-2-phenylindole (DAPI) were purchased from Cell Signaling Technology, Inc.
6
Western Blot Analysis of Glycolytic Enzymes
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Cells were lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) for protein extraction. Protein concentrations were quantified using a Pierce™ BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. A 10-μg protein was mixed with 5X SDS-PAGE loading buffer (Biosesang, Gyeonggi, Korea) and boiled at 95°C for 10 min. Boiled protein samples were separated on 10% SDS-PAGE gel and were transferred to a polyvinylidene difluoride membrane (PVDF; Sigma). Blocking of the PVDF membrane was performed using 5% skimmed milk for 1 h at room temperature. The membrane was washed with TBS-T (Tris-buffered saline with 0.1% Tween 20) three times and then incubated overnight with the primary antibody diluted in 3% bovine serum albumin (BSA) at 4°C. After the overnight incubation, the PVDF membranes were incubated with secondary anti-mouse or anti-rabbit antibodies (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h. The blots were detected using the ECL system (Pierce, Rockford, IL, USA). The primary antibodies used were anti-hypoxia-inducible factor (HIF) -1α (1:200), anti-GAPDH (1:1000), anti-HK1 (1:1000), anti-HK2 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PFKFB3 (1:1000), and anti-PFKFB4 (1:1000, Invitrogen).
7
Subcloning and Expression of SCN5A Variants
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For Western blot analysis, the expression vector pEGFP-N1 was used to subclone WT-SCN5A and A385T/R504T-SCN5A upstream of the coding region of a GFP. Western blot was performed using whole cell lysates from HEK293 cells that heterologously expressed wild type (WT), and A385T/R504T with β1-subunit SCN1B. The cells were lysated with a lysis buffer comprising 150 mM NaCl, 50 mM Tris-Cl (pH 7.4), 1 mM EDTA, and 1% Triton-X 100, and the protein concentration was determined using a BCA Protein Assay Kit (Thermo-Fisher, 23227). Next, 5X SDS-PAGE loading buffer (Biosesang, S2002) was added to the lysates, which were then separated on 8% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary antibodies, including a rabbit anti-GFP (1:1,000, Invitrogen, A-11122), and mouse anti-Na-K ATPase (1:2,000, Sigma, 05-369) overnight at 4°C, followed by a secondary antibody (1:10,000) for 1 h at room temperature. Chemiluminescence was used to detect the membrane signals, and protein expression levels were normalized to anti Na-K ATPase and quantified using ImageJ (National Institutes of Health).
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