SH-SY5Y cells were seeded in 6-well tissue culture plates at a density of 1.5 × 105 cells ml−1 and set up in CK, 4-AP, STPE-PMNSs, and STPE-PMNSs + 4-AP groups, respectively, and after 24 h of incubation, the cells were treated in the STPE-PMNSs group and STPE-PMNSs + 4-AP group with 25 μg ml−1 of STPE-PMNSs for 24 h, after which the cells were washed three times with PBS, and 4-AP was added to the STPE-PMNSs + 4-AP group for 4 h. The cells were collected, and then the cell lysates were obtained with a Whole Cell Lysis Assay (KeyGEN BioTECH Co., Ltd. Nanjing, China). The Glu and Gln contents were determined using the corresponding content assay kit. Both of their contents were determined by comparing the absorbance value with the calibration plot for standard solutions. The absorbance values were measured at 340 nm and 450 nm. All experiments were independently carried out with three replicates.
Whole cell lysis assay
Manufactured by Keygen Biotech
Sourced in China
The Whole Cell Lysis Assay is a laboratory instrument designed to facilitate the extraction and analysis of cellular contents. The device utilizes a standardized protocol to disrupt the structural integrity of cells, allowing for the isolation and examination of intracellular components such as proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (20 mentions)
Sa00001 2, by Proteintech (4 mentions)
Chemiluminescence detection system, by Bio-Rad (3 mentions)
Bca protein assay kit, by Keygen Biotech (3 mentions)
Enhanced chemi luminescence (ecl), by Keygen Biotech (3 mentions)
Ecl system, by Affinity Biosciences (3 mentions)
Bca protein quantitation assay, by Keygen Biotech (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Bca assay kit, by Keygen Biotech (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
Bicinchoninic acid assay, by Beyotime (2 mentions)
Af5145, by Affinity Biosciences (2 mentions)
Ab201060 10, by Abcam (2 mentions)
Gapdh hrp, by Proteintech (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Sds page, by Keygen Biotech (2 mentions)
44 protocols using whole cell lysis assay
1
Quantifying Glutamate and Glutamine in SH-SY5Y Cells
2
Exosomal Protein Characterization by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from the isolated exosomes or DCs from differentially treated groups using the Whole Cell Lysis Assay (KeyGen Biotech, Nanjing, China), and the concentration was determined using the BCA Protein Assay Kit (FD2001; Fude, Hangzhou, China). The lysates were resolved using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Burlington, USA).
After blocking with 5% skimmed milk in Tris-buffered saline with Tween (TBST) buffer at room temperature for 1 h, the membranes were probed with the following primary antibodies at 4°C overnight (Abcam, Cambridge, UK): TSG101 (1:1000, ab125011), CD81 (1:1000, ab109201), CD9 (1:2000, ab92726), CD63 (1:1000, ab217345), and indoleamine 2,3-dioxygenase (IDO) (1:1000, ab277522). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG H&L (1:10,000, ab205718; Abcam) and developed using BeyoECL Star (Beyotime). The intensity of the immunoreactive bands was analyzed using the ChemiDoc Imaging System (Bio-Rad, Hercules, USA).
After blocking with 5% skimmed milk in Tris-buffered saline with Tween (TBST) buffer at room temperature for 1 h, the membranes were probed with the following primary antibodies at 4°C overnight (Abcam, Cambridge, UK): TSG101 (1:1000, ab125011), CD81 (1:1000, ab109201), CD9 (1:2000, ab92726), CD63 (1:1000, ab217345), and indoleamine 2,3-dioxygenase (IDO) (1:1000, ab277522). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG H&L (1:10,000, ab205718; Abcam) and developed using BeyoECL Star (Beyotime). The intensity of the immunoreactive bands was analyzed using the ChemiDoc Imaging System (Bio-Rad, Hercules, USA).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After extensively washing, cells were lysed using Whole Cell Lysis Assay and proteins were harvested according to the protocol (KeyGen Biotech Inc., Nanjing, China). Protein concentrations was determined by the BCA Assay kit ((KeyGen Biotech Inc., Nanjing, China). Total protein of each lysate was then separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by incubated with antibodies against TLR4 (1:2,000, cat. no. 14358, Cell Signaling Technology, Inc), or IRAK4 (1:2000, cat. no. 4363, cell Signaling Technoloy, Inc), or GAPDH (1:2,000, ab8245, Abcam) overnight at 4 ℃. After washing with TBST, membranes were striped with appropriate HRP-conjugated secondary antibodies for 1h at room temperature, followed by visualized using the Luminol reagent (Thermo Fisher Scienti c, Carlsbad, CA, USA). Eventually, bands were imaged and densitometric analyzed by a chemiluminescence detection system (Bio-Rad, USA).
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from RAW264.7 cells using a Whole Cell Lysis assay (Nanjing KeyGen Biotech Co., Ltd.). Total nuclear and cytoplasmic proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd.). Protein concentration was measured using a BCA protein assay kit (Nanjing KeyGen Biotech Co., Ltd.) and 20 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% BSA (Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The membranes were then incubated overnight at 4°C with primary antibodies against NF-κB p65 (1:1,000), p-NF-κB p65 (1:1,000), Nrf2 (1:1,000), HO-1 (1:1,000), NQO1 (1:1,000), NLRC3 (1:1,000), TRAF6 (1:1,000), β-actin (1:2,000) or Histone H3 (1:2,000). Following the primary antibody incubation, the membranes were incubated with an HRP-conjugated anti-rabbit secondary antibody (Abcam; cat. no. ab6721; 1:5,000) at room temperature for 1 h. The membranes were washed multiple times with TBS-Tween-20 buffer and the protein bands were visualized using enhanced chemiluminescence reagent (cat. no. G2020; Wuhan Servicebio Technology Co., Ltd.) and a chemiluminescence imaging system (Bio-Rad Laboratories, Inc.). Densiometric analysis was performed using Image Lab software (version 6.0; Bio-Rad Laboratories, Inc.).
5
Comprehensive Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from cells using Whole Cell Lysis Assay (KeyGEN BioTECH, China) on ice and quantified by bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The western blot was performed according to standard procedures. The following primary antibodies were applied: MIF (Novus Biologicals, USA), TIMP3, PI3K, CD81, TSG101, ALIX (Abcam, USA), caspase-3, caspase-9, P-c-Raf, AKT, P-Akt (Ser473), P-Akt (Thr308), β-actin (Cell Signaling Technology, USA), BAX (Bioss ANTIBODIES, USA), P21 (Proteintech, USA), CDK2 (ImmunoWay, USA), and Cyclin H (ZenBio Science, China). The antibody information is listed in Table S2 . After primary antibody incubation, the membranes were incubated with HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies (Cell Signaling Technology, USA). The bands were captured by chemiluminescence (Biosciences, Foster City, CA, USA) through ECL detection system. Finally, the protein expression was analyzed by the software of ImageJ.
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the previous study 27 (link), protein from different groups of fibroblasts or epidural tissues were extracted using the Whole Cell Lysis Assay (Keygen, Nanjing, China). After the concentration was measured by bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA), Separation of proteins was done by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). 5% skim milk was used to seal the membrane and primary antibodies were employed to incubate the membrane at low temperature overnight. Finally, the PVDF membrane was washed by tris buffered saline-tween (TBST) for three times and incubate by secondary antibody for 1 hour at room temperature. The protein bans were visualized by electrochemiluminescence (ECL) and quantified by Image J.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using a Whole Cell Lysis Assay (KeyGEN, China), and the cytoplasmic and mitochondrial proteins were extracted using a Cytoplasmic and Mitochondrial Protein Extraction kit (Beyotime, China). The protein concentration was determined using a BCA Protein Assay kit (KeyGEN, China). Protein samples (50 μg per lane) were separated using SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, USA) for 60 min. Subsequently, the membranes were blocked with a solution containing 5% nonfat milk/bovine serum albumin for 90 min at room temperature and then treated with specific primary antibodies at 4°C overnight. Afterwards, the membranes were incubated with a fluorescent secondary antibody for 1 h at room temperature. Then, the membranes were washed three times with TBST and scanned by a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, USA). Densitometry was performed using ImageJ software.
8
Protein Expression Analysis in Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted by Whole-Cell Lysis Assay (KeyGEN BioTECH) from NRCMs and heart tissues, and the concentration was defined by the BCA Protein Assay Kit (KeyGEN BioTECH). Protein extractions were boiled and denatured, separated by 10% SDS–PAGE and then transferred onto PVDF membranes (Millipore). After blocking with 5% skimmed milk powder, the PVDF membranes were incubated overnight at 4 °C with the following rabbit-sourced primary antibodies, including anti-METTL3 antibody (Abcam, ab195352, 1:1000), anti-ANP antibody (Abcam, ab189921, 1:1000), anti-BNP antibody (Abcam, ab239510, 1:1000), anti-DKK2 antibody (Abcam, ab95274, 1:1000), anti-β-catenin antibody (Abcam, ab32572, 1:1000), anti-c-Myc antibody (Abcam, ab32072, 1:1000), and anti-β-actin antibody (ABways, AB0035, 1:5000). Subsequently, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibody (Abways, AB0101, 1:5000; AB0102, 1:2000) at room temperature for 1 h. Finally, bands were detected using ECL (KeyGEN BioTECH) with a chemiluminescence system (Bio-Rad).
9
Quantification of Liver Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from liver tissues were lysed in ice-cold Lysis buffer (Whole Cell Lysis Assay, KeyGEN, China), and the total protein concentration was quantified by a BCA Protein Assay Kit (Solarbio, China). Equal amounts of protein were electrophoresed on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% BSA, the membranes were incubated with primary antibodies (FDPS, HMGCS1 1:1,000, Proteintech) overnight at 4°C. After washing, membranes were soaked in the secondary antibodies (Anti-rabbit IgG HRP-linked, 1:5,000, Anti-mouse IgG HRP-linked, 1:5,000) for 1 h β-actin was used as an internal control. Immunoblots were then visualized by enhanced chemiluminescence (ECL) using ImageQuant LAS 5000 (GE Healthcare) and analyzed using ImageJ.
10
Whole Cell Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from cells with Whole Cell Lysis Assay (KeyGEN BioTECH, KGP2100, Jiangsu, China) on ice and quantified with bicinchoninic acid (BCA) assay kit (Beyotime, P0012, Beijing, China). Protein lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010, USA). The membranes were incubated with primary antibodies overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, MA, USA) for 1 h. Immunoreacted bands were visualized via ImageQuant LAS500 chemiluminescence (General Electric, Boston, USA) through ECL detection system (Millipore, WBKLS0500, USA). Finally, the protein expression was analyzed by the software of ImageJ (National institution of Health, USA), and β-actin was used as loading control. The primary antibodies were listed in Table S1 .
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!