For ovary lysates, ovaries were dissected and placed immediately on dry ice. Cell and ovary lysates were extracted using radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc.) plus complete protease inhibitors (Roche). Protein was quantitated using the Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific). Proteins were separated on a 4–12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to polyvinylidene fluoride membranes. Membranes were blocked in milk or Odyssey blocking buffer and incubated in primary antibodies overnight at 4°C. Primary antibodies included rabbit anti-Ref(2)P (1:10,000), mouse anti-actin (1:500; JLA20; Developmental Studies Hybridoma Bank), mouse anti-Tubulin (1:1,000; E7), mouse anti–ATPsyn-α (1:1,000), mouse anti-Porin (1:1,000; MitoSciences), mouse anti-ANT (1:500; MitoSciences), rabbit anti–Dcp-1 (1:500; Laundrie et al., 2003 (link)), guinea pig anti–Dcp-1 (1:500; Tenev et al., 2005 (link)), rabbit anti-Pink1 (1:500; Abcam), and rabbit anti-Atg8a (1:1,000; Barth et al., 2011 (link)). Membranes were incubated with HRP-conjugated secondary antibodies or infrared-labeled secondary antibodies and were detected using the ECL Enhanced Western Blotting System (GE Healthcare) or the Odyssey System (LI-COR Biosciences). Densitometry was performed using ImageQuant 5.1 software (GE Healthcare).
Complete protease inhibitors
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Complete protease inhibitors are a laboratory product designed to inhibit a broad spectrum of proteases. They are used to prevent protein degradation in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey system, by LI COR (1 mentions)
Imagequant 5, by GE Healthcare (1 mentions)
Mouse anti actin, by Developmental Studies Hybridoma Bank (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Rabbit anti pink1, by Abcam (1 mentions)
Mouse anti porin, by Abcam (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Nupage bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Heparin low molecular weight, by Santa Cruz Biotechnology (1 mentions)
5 protocols using complete protease inhibitors
1
Ovary Lysate Protein Analysis
2
Western Blot Analysis of GFP Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in pre-chilled lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1% NP-40 and one tablet of complete protease inhibitors (Santa Cruz, sc-29131) per 25 ml. Lysis was on ice for 30 min, followed by centrifugation at 14,000g, 4 °C for 10 min. Supernatant protein was quantified using Bio-Rad DC protein assay kits. Protein extracts (27 μg per lane) were resolved on 4–12% NuPAGE Bis-Tris precast gels (Life Technologies) and transferred to Bio-Rad Immun-Blot PVDF membranes (162-0177, Bio-Rad). After transfer, polyvinylidene difluoride membranes were blocked in PBS–Tween 20 (PBST, containing 1 × PBS, 0.1% Tween 20) containing 5% non-fat dry milk (M0841, LabScientific) for 1 h at room temperature, and then incubated with mouse anti-GFP primary antibody at a final concentration of 1 μg ml−1 (Santa Cruz, catalogue number SC9996,) overnight at 4 °C. Membranes were washed three times (7 min) in PBST at room temperature and incubated with secondary antibody (anti-mouse immunoglobulin G from GE healthcare, NA931V) diluted 1:4,000, for 30 min at room temperature. Subsequently, membranes were washed three times (5 min) in PBST. Peroxidase activity was measured using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) following the manufacturer's protocols; the resulting chemifluorescence was imaged using the FluorChem M imager from ProteinSimple.
3
Sperm Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly ejaculated semen samples were allowed to liquefy at 37°C for 1 h. Spermatozoa were purified using the ISolate® kit (Irvine Scientific, Santa Ana, CA, USA) followed by centrifugation for 15 min at 400 g. The pellet was resuspended in PBS, centrifuged for 10 min at 400 g and solubilized in RIPA lysis buffer in presence of complete protease inhibitors (Santa Cruz Biotechnology, Dallas, TX, USA) at a final concentration of 9 × 107 cell/mL for 30 min at 4°C. The insoluble material was pelleted by centrifugation at 10 000 g for 10 min, and the supernatant was used as the loading sample for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gels.
4
ChIP-seq protocol for Zbtb11 and GABPA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sonicated chromatin lysates were obtained as described above (see Methods: Chromatin immunoprecipitation) and the protein content was measured using the Bio-Rad RC DC protein measurement kit (cat. no. 5000121). Chromatin (500 μg) was mixed with 5 μg anti-Zbtb11 antibody overnight. Antibody/chromatin complexes were pulled down with 25 μl protein G Dynabeads by mixing for 3 h at 4 °C. The beads were washed three times and the immunoprecipitated chromatin was eluted with 50 μl [1× TE, 10 mM dithiothreitol] by shaking 30 min at 37 °C. Eluted chromatin was diluted 1:20 with 150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, Complete protease inhibitors (Roche), and mixed overnight with 8 μg anti-GABPa antibody (Santa Cruz, sc-28312 X) pre-captured on 100 μl sheep anti-mouse IgG Dynabeads (by mixing 3 h at 4 °C). Beads were washed three times and DNA was eluted with 230 μl [1× TE, 1% SDS, 1 mg/ml proteinase K, 0.3 mg/ml RNase A] by shaking overnight at 65 °C. DNA was extracted using a Qiagen PCR purification kit, and 1/40 was used in each qPCR reaction.
5
Tau Aggregation Kinetics Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monomeric human full-length Tau was aggregated by adapting a published procedure (Akoury et al., 2013) . Briefly, 50 μM of Tau were mixed with varying concentrations of heparin (0.125, 1.25, 12.5 and 125 μM) in 25 mM HEPES-KOH pH 7.5, 75 mM KCl, 75 mM NaCl, 1 mM DTT and Heparin Low Molecular Weight (Santa Cruz Biotech). Samples were heated at 95°C for 10 min and supplemented with Complete Protease Inhibitors (1/2 tablet/50 ml) and 1 mM DTT. Aggregation was performed at 37°C for shaking in an incubator at 180 rpm. Samples were collected at time points described in Fig. 4A. Monomeric human N-terminally FLAG-tagged Tau-RD was aggregated in 25 mM HEPES-KOH pH 7.5, Complete Protease Inhibitors (1/2 tablet/50 ml), 75 mM KCl, 75 mM NaCl, 10 mM DTT and Heparin Low Molecular Weight (Santa Cruz Biotech), concentrations depending on the experiment and Tau:heparin ratio were kept at 4:1. Aggregation was performed at 37°C, shaking at 180 rpm, and aliquots were flash frozen at time points indicated in the text. Aliquots were then thawed in a water bath at 37˚C for downstream applications.
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!