The SH-SY5Y cells were washed with ice-cold PBS and lysed in lysis buffer (Cwbio, CN) containing protease inhibitors cocktail (Rhoche, GER) for 30 min. The lysates were centrifuged at 12,000 × g for 10 min, and the protein concentration of the supernatants was determined with a PierceTM BCA Protein Assay Kit (Thermo, United States). A total of 20 μg of protein was electrophoresed on 8% SDS polyacrylamide gels and transferred onto PVDF membranes (300 mA, 90 min). After blocking with 10% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with rabbit anti- FBXL5 (1:1000, Abcam, United Kingdom), rabbit anti-IRP2 (1:1000, Abcam, United Kingdom), rabbit anti-TfR1 (1:1000, Abcam, United Kingdom ) and rabbit anti-β -actin monoclonal antibody (1:10000, BIOS, CN). The cross-reactivity was visualized using ECL Kit (Millopore, United States) and analyzed through scanning densitometry with a UVP image system.
Lysis buffer
Manufactured by CWBIO
Sourced in China
Lysis buffer is a solution used in molecular biology and biochemistry to disrupt cells and extract cellular components, such as proteins, nucleic acids, or organelles. It typically contains a combination of detergents, salts, and other agents that facilitate the breakdown of cell membranes and the release of the desired cellular contents.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Rabbit anti irp2, by Abcam (1 mentions)
Rabbit anti tfr1, by Abcam (1 mentions)
Runx2, by Abcam (1 mentions)
Imagej, by Techcomp Instruments (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Eecl kit, by CWBIO (1 mentions)
Protease inhibitor cocktail, by CWBIO (1 mentions)
Phosphatase inhibitor, by CWBIO (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
Protease inhibitor, by CWBIO (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using lysis buffer
1
Protein Analysis of SH-SY5Y Cells
2
Protein Expression Analysis of hASCs
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The proteins from hASCs from the experimental and control groups at day 7, day 14, and day 21 were extracted using the cell protein extraction kit. The cells in each dish were lysed in 100 mL lysis buffer (CWBIO, Beijing, China) containing 1 mM phosphorylated protease inhibitor (Thermo, USA), and the samples were incubated on ice for 20 minutes. The extracted proteins were detected by BCA analysis and were loaded on 10% or 12% SDS-PAGE gel electrophoresis. The samples were transferred to a PVDF membrane and incubated with blocking solution of TBST containing 0.05 g/mL bovine serum albumin (BSA) for one hour. After blocking, the samples were incubated with their corresponding primary antibodies overnight at 4°C. The primary antibodies included OPN (1: 2,000, Abcam), OCN (1: 1,000, Abcam), and RUNX-2 (1: 1,000, Abcam). After washing the membrane three times in TBST, the membrane was incubated with the corresponding secondary antibody for one hour. After washing the membrane three times in TBST, the immunoreactive band was detected with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The optical densities of the bands were quantified using ImageJ (Scion Corporation).
3
Western Blot Analysis of IRAK1, TRAF6, and pNF-κB in Rat Dorsal Root Ganglia
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Total proteins from rat L4-L6 DRGs or SDH were extracted with lysis buffer (CWBio, Beijing, China). Briefly, 30 μg of each sample was resolved through sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto Immobilon-P polyvinylidene difluoride (GE). After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with an anti-IRAK1 antibody, anti-TRAF6 antibody, anti-pNF-κB (p65) antibody, and anti-β-actin antibody. The corresponding secondary antibodies were probed after washing the membranes. Final results were acquired using a western blot detection system (GE) with enhanced chemiluminescence reagents eECL Kit (CWBio, Beijing, China). Table 3 lists the primary and secondary antibodies used for the western blot analysis.
List of primary and secondary antibodies used for western blot analysis
| Antibody | Host | Company | Catalog number | Dilution | Incubation conditions |
|---|---|---|---|---|---|
| IRAK1 | Rabbit | Abcam | ab238 | 1:1000 | Overnight 4 °C |
| TRAF6 | Rabbit | Abcam | ab181622 | 1:1000 | Overnight 4 °C |
| pNF-κB (p65) | Mouse | Cell signaling technology | #13346 | 1:1000 | Overnight 4 °C |
| β-actin | Mouse | ZSGB-BIO | TA-09 | 1:1000 | Overnight 4 °C |
| Anti-rabbit IgG horseradish peroxidase (HRP) | Goat | ZSGB-BIO | ZDR-5306 | 1:3000 | 1 h RT |
| Anti-mouse IgG horseradish peroxidase (HRP) | Goat | ZSGB-BIO | ZDR-5307 | 1:3000 | 1 h RT |
4
Western Blot Analysis of BASP1 and GAPDH
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Cells were washed with icy phosphate-buffered saline (PBS, Pleasanton, CA, USA) and lysed with lysis buffer (CWBIO, Beijing, China) containing 1% protease inhibitor Cocktail (CWBIO) and 1% phosphatase inhibitors (CWBIO). All protein lysates were centrifuged with 10000g at 4°C for 15 min to remove cell precipitates. Protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, loading buffer was added to the proteins and cooked at 99°C for denaturation. Proteins were loaded on a 10% SDS PAGE Gel and transferred onto a PVDF membrane (Millipore, Boston, MA, USA). After being blocked with milk, the membranes were incubated with indicated primary antibody at 4°C overnight. The primary antibodies were listed as follows: BASP1 (Bioss, Beijing, China, 1:1000) and GAPDH (Proteintech, Rosemont, IL, USA, 1:2000). The HRP-linked secondary antibody (CST, Danvers, MA, USA 1:3000) was incubated at room temperature for one hour. The chemiluminescence signals were detected by ChemiDoc Touch Imaging System (Bio-Rad, Berkeley, CA, USA).
5
Locust Brain Protein Analysis
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Locust brains (8–10 individuals/sample) were collected and homogenized in lysis buffer (CWBIO) containing protease inhibitor (CWBIO). The total protein content was determined using a bicinchoninic acid protein assay kit (Thermo Scientific). The extracts were reduced, denatured, and separated by gel electrophoresis on a 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated separately with specific antibodies against Piwi1, Piwi2, and Ago3 (mouse anti‐Piwi1 serum, 1:500; mouse anti‐Piwi2 serum, 1:500; and mouse anti‐Ago3 serum, 1:500, respectively), hnRNP F/H (Mouse monoclonal antibody, 1:2,000, Abcam), and U2AF65 (Mouse monoclonal antibody, 1:200, Santa Cruz Biotechnology). Tubulin was used as an endogenous control (rabbit polyclonal antibody, 1:5,000, CWBIO). Goat anti‐rabbit IgG was used as the secondary antibody (1:10,000, CWBIO). The intensities of the Western blot bands were quantified using densitometry with Quantity One software.
6
Sichong Formula Modulates Gastric Cancer
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After treated with 80 μg/mL Sichong formula for 48 hrs, gastric cancer cells were lysed by aradio-immunoprecipitation assay Lysis Buffer (CWBIO, Beijing, China) added with 1% protease inhibitor. A BCA method (Beyotime, Beijing, China) was used for the quantification of protein concentration. 20μg of total proteins was taken out and subjected to 10% SDS-PAGE electrophoresis. The protein bands were then electroblotted onto apolyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking in TBST buffer (TBS buffer with 0.1% Tween-20) containing 5% BSA for 1 hr at room temperature, protein bands were successively incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 hr at room temperature. Then, protein bands were washed by TBST buffer for three times every 5 mins and visualized using Immobilon western (Millipore Co.). The primary antibodies against Bcl2, Bax, C-Caspase 3, α-tubulin, LC-3, Beclin1, P62, AKT, p-AKT, and Cyclin D1 were purchased from Cell Signaling Technology (Beverly, MA, USA). All secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).
7
Quantitative Protein Analysis in Peri-Hemorrhage Brain
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Total protein was extracted from isolated peri-hemorrhage brain tissue by homogenization with lysis buffer (CWBIO, China) and protease inhibitor. The tubes were kept for 30 minutes on ice before being subjected to 15 minutes of centrifugation at 14000×g at 4 °C. The Micro BCA Protein Assay (Thermo Fisher Scientific Inc., MA, USA) was used to quantify protein concentrations. The extracted proteins were then electrophoresed with 10% SDS-PAGE gel and blotted onto Poly Vinylidene Difluoride (PVDF) membranes (Millipore, MA, USA). These membranes were immersed in 5% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) for 1 h to block endogenous reactions and incubated overnight with primary at 4 °C. Secondary antibodies were then added the next morning at room temperature for 1 h. The membranes were probed with specific primary antibodies and HRP-conjugated secondary antibodies. The blots were visualized with ECL luminescence reagents.
8
Quantitative Western Blot Analysis
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Total protein was extracted from the cell cultures according to instructions from radioimmunoprecipitation assay Lysis Buffer (CWBIO Tech). Protein concentration was quantified using an Enhanced Bicinchoninic Acid Protein Assay kit. A total of 20 µg protein from each sample were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were incubated in blocking buffer [5% non-fat milk dissolved in TBS with Tween-20 (TBST)] at room temperature for 1-h and then probed at 4°C overnight with primary antibodies (dilution, 1:1,000). The membranes were subsequently rinsed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1-h at room temperature. The blots were visualized using the enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) and analyzed using the Quantity One 1-D software v4.6.7 (Bio-Rad Laboratories, Inc.). Each experiment was repeated at least three times.
9
Western Blotting Analysis of DNA Damage Signaling
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To determine the protein expression, western blotting was performed. MDA-MB-231 cells were lysed on ice in lysis buffer (Cwbiotech, Beijing, China) including protease inhibitor cocktail. The total protein was quantified with BCA Protein Assay Kit as described previously [21]. Antibodies, anti-p-BRCA1 (Ser1524), anti-53BP1, anti-p-ATM(Ser1981), anti-p-p53(Ser15), anti-p53, anti-p-CHK1(Ser317), anti-p-Stat3(Tyr 705), anti- Stat3, anti-β-actin, anti-β-tubulin and anti-GADPH.
10
Western Blot Analysis of Mitochondrial and Lipid Metabolism Proteins
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Samples were fragmented and lysed in lysis buffer at 4 °C for 30 minutes (CWBIO, China). After centrifugation at 12 000×g at 4 °C for 30 minutes, the supernatants were collected, followed by quantification of sample protein concentrations using a BCA Protein Quantitative Assay Kit (CWBIO). Following mixing with loading buffer and boiling for denaturation, protein mixtures were loaded into each well and separated on an 8% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gel. Proteins were then transferred onto nitrocellulose membranes and blocked for 2 hours. After incubation with rabbit polyclonal antibodies against proteins related to mitochondrial dynamics and lipid metabolism at 1:1000 dilution at 4 °C overnight, the membranes were washed with tris‐buffered saline with Tween 3 times and incubated with the secondary antibody for 90 minutes, followed by washing with tris‐buffered saline with Tween 3 times. Protein bands were detected with ECL Western blot detection kits (CWBIO), and their levels were quantified using ImageJ. All experiments were performed at least 3 times, and the averages were used for comparisons.
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