Following exposure to CM (0, 0.5, 1, 2 µmol/l for PRMI8226 cells, and 0, 2, 4, 8 µmol/l for U266 cells) for 48 h, the cells were harvested and washed with ice-cold PBS. Proteins were extracted using ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) with phenylmethylsulfonyl fluoride. Following a brief sonication, the cell lysates were kept on ice and subsequently centrifuged at 15,984 × g for 10 min. Supernatant was collected and the protein concentrations were quantified with a bicinchoninic acid assay (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Total protein (40 µg/lane) was separated using 10–12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was subsequently blocked with TBST (0.05% Tween-20) containing 5% skimmed milk for 1 h and probed overnight with the corresponding primary antibodies (diluted in primary antibody dilution buffer from Beyotime Institute of Biotechnology) at 4°C. Subsequently the membrane was washed with TBST, exposed to the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature, and detected by enhanced chemiluminescence (EMD Millipore).
Primary antibody dilution buffer
Manufactured by Beyotime
Sourced in China, United States, United Kingdom
Primary antibody dilution buffer is a solution used to dilute primary antibodies for immunoassays. It helps maintain the stability and specificity of the primary antibody during the dilution process.
Automatically generated - may contain errors
Lab products found in correlation
Secondary antibody dilution buffer, by Beyotime (12 mentions)
Ecl plus, by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Ripa buffer, by Beyotime (5 mentions)
Beyoecl plus, by Beyotime (4 mentions)
Bradford protein assay kit, by Beyotime (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (4 mentions)
Sds loading buffer, by Beyotime (4 mentions)
Ecl plus reagent, by Solarbio (4 mentions)
Anti cdk2, by Sangon (4 mentions)
Anti pcna, by Sangon (4 mentions)
Anti acvr1, by Abcam (4 mentions)
Anti myod, by Abcam (4 mentions)
Anti myog, by Abcam (4 mentions)
Bca protein assay kit, by MultiSciences Biotech (4 mentions)
Anti cyclin d1, by Abcam (4 mentions)
77 protocols using primary antibody dilution buffer
1
Western Blot Analysis of PRMI8226 and U266 Cells
2
Quercetin and Rutin Cytotoxicity Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quercetin, rutin, dimethyl sulfoxide (DMSO) and 5-fluorouracil were purchased from Shanghai Kaiyang Biotechnology Company (Shanghai, China). Acetonitrile (HPLC), RPMI Medium 1640, and fetal bovine serum (FBS) were products of Gibco Life Technologies (NY, USA). The Apoptosis and Necrosis Assay Kit was purchased from the Beyotime; OLYMPUS IX73. Guava easyGyte; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), Cell lysis buffer for Western and IP, the SDS-PAGE Gel Quick Preparation Kit, Protein Marker, Polyvinylidene Fluoride (PVDF) Membrane, Blocking Buffer, Primary Antibody Dilution Buffer, Secondary Antibody Dilution Buffer, and BeyoECL Plus were purchased from the Beyotime Institute of Biotechnology (Shanghai, China).
3
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 protein was lysed in RIPA (Thermo Fisher Scientific) along with protease and phosphatase inhibitor cocktail, quantified by BCA Assay. Protein samples were separated on non-reducing SDS-PAGE 12% Tris–HCl gels (Beyotime, Shanghai, China) and then transferred onto PVDF membranes. The membrane was then blocked in 5% skimmed milk for 1 h, after which the membrane was incubated with primary antibodies diluted in primary antibody dilution buffer (Beyotime, Shanghai, China) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Blots were developed with chemiluminescent detection reagent and imaged with a Chemiluminescent Imaging System (Tanon, Shanghai, China). Western blot quantification was conducted using ImageJ. The western blot antibodies, were listed as follows: anti-PRLR (Abcam, ab170935, 1: 1,000), anti-TNFSF13B (Abcam, ab224710, 1: 1,000), anti-GAPDH (Cell Signaling Technology, 5174, Massachusetts, USA, 1: 1,000), anti-NF-κB p65 (Cell Signaling Technology, 8242, 1: 1,000), anti-Phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, 3033, 1: 1,000), anti-rabbit IgG (Cell Signaling Technology, 7074, 1: 5,000 in Tris Buffered Saline-Tween).
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were first rinsed in ice-cold phosphate-buffered saline (PBS), pH7.2, and lysed in 1% NP-40, 125 mM Tris, pH6.8, containing a protease inhibitor cocktail (Roche, Basel, Switzerland). For western blotting, 35–50 µg protein was added per lane of 12% SDS-PAGE. Primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotech., Shanghai, China). Antibodies used included: anti-GAPDH (Abgent Biotech. Co. Ltd., Suzhou, China), anti-ferritin, SOD1, SOD2, SDHB and ISCU (Abcam, Cambridge, MA, USA), anti-catalase (Santa Cruz Biotech., Santa Cruz, CA, USA), anti-XOD antibody (ProteinTech Inc., Chicago, IN, USA), anti-TfR1 antibody (Zymed, San Francisco, CA, USA), anti-FXN, IRP1, and IRP2 (polyclonal, raised from rabbit). Detection was performed using peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, UK). Quantification of the density of the western bands was done with programme ImageJ (http://rsb.info.nih.gov/ij/ ).
5
Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer with 1% phenylmethylsulfonyl fluoride and 1% protein phosphatase inhibitor (Beyotime, China) on ice for 20 min. Proteins in the mouse tissue homogenate were extracted with RIPA buffer (Beyotime, China). Lysates were centrifuged at 12,000 rpm for 30 min at 4°C to obtain supernatants. Equal amounts of protein lysates in RIPA were separated on 8%, 10% or 12% SDS-PAGE gels before being transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). The membranes were blocked with 5% milk and then incubated with primary antibodies in primary antibody dilution buffer (Beyotime, China) at 4°C overnight. The blots were probed with the following antibodies: Tau 1:1,000, P-Tau Thr181 1:1,000, Akt 1:1,000, P-Akt Ser 473 1:2,000, P-ERK1/2 Thr202/Tyr204 1:2,000, ERK1/2 1:1,000, Caspase 3 1:1,000, P-PKCζ/λ Thr410/403 1:1,000 (Cell Signal Technology, USA); KIBRA 1:500, PKCζ 1:1,000 (Santa Cruz, USA); PARP-1 1:1,000 (Abcam, UK); β-actin 1:2,000 (Proteintech, China). The membranes were subsequently incubated with peroxidase-conjugated secondary antibodies and developed using the enhanced chemiluminescence (Merck Millipore) method. Band densities were quantified by densitometry using Fluor ChemQ software.
6
Proteomic Analysis of Fetal Bovine Serum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fetal bovine serum (KGY009), incomplete culture medium (KGM1640SF), and the Nuclei and Cytoplasm Protein Extraction Kit (KGP150) were supplied by KeyGen Biotech (Nanjing, China). The Bradford protein assay kit, Cell Counting Kit-8 (CCK-8 Kit), western blocking buffer, primary antibody dilution buffer, and secondary antibody dilution buffer were from Beyotime Institute of Biotechnology (Haimen, China). Trypsin was from Promega (Madison, USA). Nonlinear immobilized pH gradient (IPG) strips were purchased from GE (Piscataway, USA). Chemicals used for 2DE were purchased from Amresco (Solon, USA). ZnSO4∙7H2O was purchased from Aladdin (Shanghai, China). All water used in experiments was Millipore Milli-Q filtered at a resistivity greater than or equal to 18.25 MΩ·cm. Culture dishes, and polyvinylidene difluoride (PVDF) membranes were from Millipore (Bedford, USA). Primary antibodies were purchased from the following vendors: anti-actin antibody (AA128, Beyotime); Hsp90α (D1A7) rabbit mAb (CST#8165) and hnRNPA1 (D21H11) rabbit mAb (CST#8443) (both Cell Signaling Technology). Secondary antibodies, including goat anti-rabbit lgG-HRP (sc-2004) and goat anti-mouse lgG-HRP (sc-2005), were purchased from Santa Cruz Biotechnology.
7
Western Blot Analysis of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates of lung tissue were prepared. Then, the proteins were separated by 10-12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% skim milk for 1 h RT, follow by incubated with primary antibodies overnight at 4 ℃. The following day, membranes were washed and incubated with HRP-conjugated secondary antibodies for 1 h and proteins were visualized using the ECL reagent. Rabbit anti-CitH3 (ab5103, 1:1000) was purchased from Abcam; Mouse anti-MPO (AF3667, 1:400) was purchased from R&D; beta (β)-Tubulin (66,240–1-Ig, 1:5000) was purchased from Proteintech Technology. All primary antibodies were diluted in primary antibody dilution buffer (Beyotime Biotechnology, China), and secondary antibodies were diluted in 5% skim milk.
8
Fluorescence Microscopy Cell Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For fluorescence microscopy procedures, cells were seeded on circular glass coverslips (WHB Scientific, #WHB-12-CS-LC) placed in 12-well plates. Cells, transfected with expression vectors for (fluorescent)-tagged proteins or used in OMV endocytosis or gentamicin protection assays, were washed with PBS and fixed with 4% paraformaldehyde (Biosharp, #BL539A) for 20 min at room temperature. Cells were subsequently incubated with QuickBlock blocking and permeabilization buffer (Beyotime, #P0260) for 40 min before staining for 12 h at 4 °C with primary antibodies diluted in primary antibody dilution buffer (Beyotime, #P0262). Finally, slides were incubated for 2 h at 25 °C with secondary antibodies diluted in secondary antibody dilution buffer (Beyotime, #P0265). For nuclear staining, 10 μg/mL DAPI solution (Solarbio, #C0065) was used for 15 min at room temperature. Images were acquired on Zeiss LSM880 or Olympus FV3000 microscopes.
9
Anticancer Effects of UNC0638 Compound
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UNC0638 was purchased from MedchemExpress LLC (Princeton, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and low melt point soft agar were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Invasion chambers and matrigel were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Gentian violet was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kits (cat. no. 556570) were purchased from BD Biosciences. Cell lysis buffer (cat. No. P0013), a BCA protein assay kit (cat. no. P0011) and Primary Antibody Dilution Buffer (cat. no. P0023A) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Polyvinylidene fluoride (PVDF) membrane and chemiluminescent reagents were obtained from EMD Millipore (Billerica, MA, USA). Antibodies to E-cadherin (20874–1-AP), vimentin (10366–1-AP), Slug (12129–1-AP), Snail (13099–1-AP) and β-actin (60008–1-Ig) were purchased from Proteintech Group, Inc. (Chicago, IL, USA) and diluted at 1:1,000 with Primary Antibody Dilution Buffer (Beyotime, Beijing, China).
10
Quantification of Protein Expression Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein sample was obtained from 0.1 g tissue samples with 1,000 μL RIPA lysis buffer containing 10 μL phenylmethanesulfonyl fluoride. The target proteins were isolated with SDS-PAGE and transferred onto PVDF membranes. Next, the membranes were sealed with 5% skim milk, and following primary antibodies were incubated overnight in a refrigerator (4℃): β-tubulin (1:1,800), β-actin (1:3,000), mTOR (1:600), ATG5 (1:1,000), and LC3B (1:1,000) from Abmart (Shanghai, China); Nrf2 (1:1,000) from Bioss (Beijing, China); Keap1 (1:1,800), GPx-1 (1:1,000), NQO1 (1:1,000), HO-1 (1:1,000), and p62 (1:800) from Wanleibio (Shenyang, China); and Beclin 1 (1:1,200) from ABclonal (Wuhan, China), all diluted in primary antibody dilution buffer (Beyotime, Shanghai, China). The membrane was incubated with the secondary goat anti-mouse antibody (1:5,000) and anti-rabbit IgG antibody (1:8,000), both from Bioss, for 1 h at 28℃. Lastly, the protein was identified using a hypersensitive chemiluminescence kit (Beyotime), and scanning and imaging was performed using a gel imaging system. Densitometric analysis of the protein bands was analyzed with ImageJ (Version 1.38) (Ali Shah et al., 2021 (link)).
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!