RPMI-1640, fetal bovine serum (FBS), glutamine, penicillin, streptomycin, and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid were purchased from Gibco (Grand Island, NY, USA). Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China). FK506, MFN2, and β-actin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Methyl-thiazolyl-tetrazolium (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-2, IL-4 and interferon (IFN)-γ were obtained from Biosource (Worcester, MA, USA). Fluo-3/AM and pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Total RNA isolation and reverse transcription systems were purchased from Promega (Madison, WI, USA). The Biomol Green Calcineurin Assay kit was purchased from Biomol (Plymouth Meeting, PA, USA). Nuclear extract and TransAM NFAT kits were obtained from Active Motif (Carlsbad, CA, USA). Nondenaturing lysis buffer and protease inhibitor cocktail were purchased from Applygen Technologies Inc., (Beijing, China). An Amersham enhanced chemiluminescence (ECL) Advance Western Blotting Detection kit was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).
Nondenaturing lysis buffer
Manufactured by Applygen
Sourced in China
Nondenaturing lysis buffer is a laboratory reagent used to extract proteins from cells or tissues while maintaining their native structure and function. It is designed to gently disrupt cell membranes without denaturing the proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
β actin primary antibody, by Santa Cruz Biotechnology (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Applygen (1 mentions)
Phorbol myristate acetate (pma), by Beyotime (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti p stat1, by Cell Signaling Technology (1 mentions)
Anti cx3cr1, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Mitochondria cytosol fractionation kit, by Applygen (1 mentions)
8 protocols using nondenaturing lysis buffer
1
Immunomodulatory Effects of Calcineurin Inhibition
2
Western Blot Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary Abs were used: anti-CD49b, anti-STAT-1, anti-pSTAT-1 (Cell Signalling Technology Inc., Danvers, MA, USA), anti-CX3CL1 (eBioscience, San Diego, CA, USA), anti-CX3CR1 (eBioscience), anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-GAPDH (Hangzhou Goodhere Biotechnology Co. Ltd, Hangzhou, China). Proteins were extracted by nondenaturing lysis buffer (Applygen, Beijing, China), and the concentration was determined by a bicinchoninic acid Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins were separated by SDS–PAGE and transferred onto a nitrocellulose membrane (Pall, New York, NY, USA). The membranes were blocked in 5% skimmed dry milk in TBST at 37 °C for 1 h and then incubated with primary Abs at 4 °C overnight, followed by incubation with secondary Abs conjugated to HRP at 37 °C for 1 h (KPL, Gaithersburg, MD, USA). Chemiluminescence reactions were performed with an ECL Detection Kit (Pierce), and images were acquired using a Kodak X-Omat film (Carestream, Xiamen, China). Bands were analysed using Bio-Rad Quantity One software (Bio-Rad, Hercules, CA, USA), and expression was calculated as the ratio of the signal for the specific protein to the signal for actin or GAPDH.
3
Immunoprecipitation of Cysteine Adducts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated at a density of 5 × 106 cells/dish and cultured for 24 h. Then the cells were treated with SFN-Cys (30 μM) and SFN-NAC (30 μM) for 24 h, and washed with ice-cold PBS, then lysed on ice via Nondenaturing Lysis Buffer (APPLYGEN, China) with protease inhibitors cocktail. The cell lysates were incubated with the corresponding antibody overnight at 4 °C. The complexes were pulled down with protein A/G agarose for 3 h and the proteins were isolated by centrifuging and boiling for 5 min. Western blot was used to recognize the conjugated proteins.
4
Protein Extraction and Purification for α-Tubulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with 30 μM SFN-NAC for 24 h and then lysed with Non-denaturing Lysis Buffer (Applygen) for 30 min on ice and the protein was extracted by centrifugation at 12,000 g for 30 min. The anti-α-tubulin (Ruiying Biological) was added to the protein lysate, incubated overnight at 4°C. The α-tubulin complexes were pulled down with protein A/G agarose for 3 h, and the proteins were purified by centrifugation and boiled for 5 min to reverse the crosslink. Western blot was used to recognize the conjugated proteins.
5
Mitochondrial Fractionation and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with CHB as mentioned earlier, KB and KBV200 cells were collected and resuspended in Mito-Cyto extraction buffer provided by the Mitochondria/Cytosol Fractionation Kit (Applygen Technologies, Beijing, People’s Republic of China), and kept on ice for 30 minutes. The cell suspension was added to a Dounce homogenizer and homogenized for 30 strokes on ice. The cell lysate was then centrifuged at 800× g for 5 minutes at 4°C to pellet the nucleus and cell debris. The supernatant was collected and centrifuged at 800× g for 5 minutes at 4°C again to thoroughly pellet the nucleus and cell debris. The supernatant was collected and centrifuged at 12,000× g for 10 minutes at 4°C to pellet mitochondria. Mitochondria were kept in an ice bath during the isolation process. The supernatant containing cytosol protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The mitochondrial pellet was lysed in nondenaturing lysis buffer (Applygen Technologies Inc.) for 30 minutes on ice, centrifuged at 12,000× g for 15 minutes at 4°C. The supernatant containing mitochondrial protein was analyzed by SDS-PAGE.
6
PDGFR-β Signaling in U-CH2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected U-CH2 cells were lysed in nondenaturing lysis buffer (Applygen). For Western blotting, protein samples (20 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. Different blots were incubated with antibodies against PDGFR-b (1:1000, Cell Signaling Technology), phospho-PDGFR-b antibody (1:1000, Abcam), mammalian target of rapamycin (mTOR) antibody (1:100, Cell Signaling Technology), or phospho-mTOR antibody (1:1000, Abcam) and GAPDH (1:5000, Topunive), followed by secondary antibodies tagged with horseradish peroxidase (Santa Cruz Biotechnology). Blots were visualized by enhanced chemiluminescence, and densitometry was performed with an imaging apparatus (Amersham Imager 600, GE). Analysis of GAPDH levels was used as a loading control.
The mRNA expression level of PDGFR-b was measured using quantitative reverse transcription polymerase chain reaction. The first-strand cDNA was synthesized from total RNA (800 ng) using the SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative reverse transcription polymerase chain reaction was performed using an ABI 7500 Fast system (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems). The primers were as follows: CGTCAAGATGCTT A AATCCACAGC (forward) and TGATGATATAGAT G G GTCCTCCTTTG (reverse).
The mRNA expression level of PDGFR-b was measured using quantitative reverse transcription polymerase chain reaction. The first-strand cDNA was synthesized from total RNA (800 ng) using the SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative reverse transcription polymerase chain reaction was performed using an ABI 7500 Fast system (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems). The primers were as follows: CGTCAAGATGCTT A AATCCACAGC (forward) and TGATGATATAGAT G G GTCCTCCTTTG (reverse).
7
Uterine Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice uterine proteins were extracted with non‐denaturing lysis buffer (Applygen, Beijing, China), and the protein concentration was examined using a Bicinchoninic Acid Protein Assay Kit (Pierce, Rockford, IL, USA). Uterine proteins were separated by 10% SDS‐PAGE and transferred onto a nitrocellulose membrane (Pall, New York, NY, USA). The membranes were blocked with 10% bovine serum albumin (BSA) in TBST at 37°C for 1 hr and then incubated with the primary antibodies at 4°C overnight. The following primary antibodies were used: rabbit anti‐CD49b (ab133557; Abcam, Cambridge, UK) and rabbit anti‐GAPDH (Boster, Wuhan, China). The membranes were washed and incubated with goat anti‐rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (KPL, Gaithersburg, MD, USA) at 37°C for 1 hr. Then, the membranes were washed. Chemiluminescence reactions were performed with an ECL Detection Kit (Pierce), and images were acquired using Kodak X‐Omat film (Carestream, Xiamen, China). Relative protein levels were analysed using Bio‐Rad Quantity One software (Bio‐Rad, Hercules, CA, USA) and were normalized to GAPDH.
8
Immunoprecipitation and Immunoblotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in Nondenaturinglysis buffer (C1050, Applygen, Beijing, China). Cell lysates were separately incubated with a specific primary antibody, including anti-p62 (PM045, MBL), anti-LC3B (M186-3, MBL) and anti-insulin (L6B10, CST), overnight at 4°C; then with protein A/G-Sepharose (Santa Cruz Biotechnology) for 3 h. The immunoprecipitated complexes were collected by centrifugation at 4°C, 3000 rpm for 3 min and then eluted 5 times by the lysis buffer. The eluted protein samples were separated on SDS-PAGE and followed by immunoblotting using the three antibodies mentioned above, anti-LAMP2 (L0668) and anti-β-actin (A5441) (both from Sigma-Aldrich US).
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!