Protease inhibitor

Manufactured by New England Biolabs

Protease inhibitor is a laboratory product designed to prevent the degradation of proteins by proteolytic enzymes. It functions by inhibiting the activity of proteases, enzymes that break down proteins. This product helps maintain the integrity of protein samples during experimental procedures.

Automatically generated - may contain errors

Lab products found in correlation

Rnase inhibitor, by New England Biolabs (2 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Ribonucleoside vanadyl complex, by New England Biolabs (1 mentions) Rnase inhibitor, by Takara Bio (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Anti brg1, by Santa Cruz Biotechnology (1 mentions) Anti myod, by Santa Cruz Biotechnology (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Flag m2 magnetic beads, by Merck Group (1 mentions) Rnaseout, by New England Biolabs (1 mentions) Dnase 1, by New England Biolabs (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Murine rnase inhibitor, by New England Biolabs (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Protein a g plus agarose beads, by Thermo Fisher Scientific (1 mentions) Anti hnrnp h, by Fortis Life Sciences (1 mentions)

Spelling variants of this product

Protease inhibitor cocktail (13 citations) Protease/phosphatase inhibitors (3 citations) EDTA-free Protease Inhibitor (2 citations) Protease inhibitor Cocktail EDTA-Free, (2 citations)

6 protocols using protease inhibitor

Subcellular Fractionation for RNA and Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols

Two million cells were resuspended in 250 μl of fractionation buffer (50 mM Tris–HCl, pH 7.6, 50 or 200 mM KCl, 5 mM MgCl₂, 0.1% NP40, 1 mM DTT, 100 units/ml RNase inhibitor (NEB, #M0314), 1× protease inhibitor cocktail), incubated on ice for 10 min, and centrifuged at 2000 g for 2 min to remove nuclei. Cell lysates were transferred to new Eppendorf tubes and centrifuged again. After 40 μl of the lysate was taken out for total cytoplasmic lysate, 200 μl of the lysate was centrifuged at 12 000 g for 15 min. The upper fraction was saved as the supernatant. Fractionation buffer was used to wash the gel-like precipitate. Care was taken not to disturb the precipitate when removing the buffer. After a second wash, the precipitate was resuspended in Trizol for RNA or 8 M urea for protein analysis.

Jin S.W., Seong Y., Yoon D., Kwon Y.S, & Song H. (2024). Dissolution of ribonucleoprotein condensates by the embryonic stem cell protein L1TD1. Nucleic Acids Research, 52(6), 3310-3326.

+ Open protocol

+ Expand

Immunoprecipitation and Western Blot Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Calbiochem, La Jolla, CA). Protein concentrations in extracts were measured using a bicinchoninic acid assay (Pierce). A volume of extract containing 200 μg protein was immunoprecipitated, subjected to SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidenedifluoride membranes. For RNA immunoprecipitation assays, RNase Inhibitor (40 U μl⁻¹; TaKaRa) and protease inhibitor were added to the cell lysis buffer, and ribonucleoside–vanadyl complex (10 mM; New England BioLabs) was added to the wash buffer. Antibodies specific to Baf60c were a gift from Dr Pier Lorenzo Puri (Forcales, 2012); anti-MyoD (Santa Cruz, SC-760) and anti-Brg1 (Santa Cruz, sc-17796X) were obtained commercially. Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Yu X., Zhang Y., Li T., Ma Z., Jia H., Chen Q., Zhao Y., Zhai L., Zhong R., Li C., Zou X., Meng J., Chen A.K., Puri P.L., Chen M, & Zhu D. (2017). Long non-coding RNA Linc-RAM enhances myogenic differentiation by interacting with MyoD. Nature Communications, 8, 14016.

+ Open protocol

+ Expand

Zebrafish Dicer1 Overexpression and miR-451 Pulldown

Check if the same lab product or an alternative is used in the 5 most similar protocols

mRNAs encoding for FLAG-tagged DrDicer1 was injected into single-cell stage zebrafish embryos (1 nL of 0.1 μg/μL stock) together with pre-miR-451 (1 nL of 10 μM stock). 100 embryos were collected 7 h after injection from each sample and washed twice with 1X PBS. The embryos were lysed with 500 μL of NET-2 buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40) and 1 μL of RNaseOUT (New England Biolabs) and protease inhibitor (Roche). Lysates were cleared by centrifugation at 16,000 x g. 50 μL of supernatant was kept as input and the rest of the was added to 500 μL of NET-2 with 50 μL of FLAG M2 magnetic beads (Sigma) and incubated for 2 h at 4°C. 50 μL of supernatant was removed for further analysis. The beads were washed 3 times with 500 μL of NET-2 buffer. 500 μL of Trizol (Invitrogen) was added to the beads to extract total RNA.

Kretov D.A., Walawalkar I.A., Mora-Martin A., Shafik A.M., Moxon S, & Cifuentes D. (2020). Ago2-dependent processing allows miR-451 to evade the global microRNA turnover elicited during erythropoiesis. Molecular cell, 78(2), 317-328.e6.

+ Open protocol

+ Expand

Protein Extraction and Fractionation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Samples were collected after 3 and 20 h of induction, and equivalent number of cells were pelleted down and re-suspended in 0.3 ml of lysis buffer (20 mM Tris pH 8500 mM NaCl, 0.2 mg/ml lysozyme) supplemented with DNAseI (NEB) and protease inhibitors (Roche). The samples were incubated on ice for 30’, sonicated, and then were spun down at 14 000 rpm for 10’ at 4°C. The supernatant was collected (soluble fraction), whereas the pellet was washed twice in PBS and then re-suspended in 0.3 ml of PBS (insoluble fraction). After adding equal volume of 2xSDS-PAGE loading buffer, the fractions were boiled for 10’ at 100°C and ran on a 4–20% SDS-PAGE gel (NuPAGE).

Morra R., Shankar J., Robinson C.J., Halliwell S., Butler L., Upton M., Hay S., Micklefield J, & Dixon N. (2015). Dual transcriptional-translational cascade permits cellular level tuneable expression control. Nucleic Acids Research, 44(3), e21.

+ Open protocol

+ Expand

Rapid Cytoplasmic Protein Extraction

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were collected using trypsin–EDTA (0.05%, Thermo), washed with 1× PBS, and incubated in a hypotonic buffer (30 mM HEPES–KOH, pH 7.4; 100 mM potassium acetate; 2 mM Mg(OAc)₂; 2 mM NaF; 5 mM DTT, 0.1% NP-40) supplemented with murine RNase inhibitor (5 U/100 μl (NEB)) and protease inhibitors (Roche) for 10 min on ice. Cells were broken by passage (20×) through a hypodermic needle (26 G). Intact cells were discarded after centrifugation at 300 g for 7 min at 4°C, followed by removal of nuclei through centrifugation at 1000 g for 10 min at 4°C. The concentration of the cytoplasmic protein extracts was measured using Bradford reagent (Biorad), followed by snap-freezing aliquots (10 μg) in liquid nitrogen and storage at -80°C until use.

Drino A., König L., Capitanchik C., Sanadgol N., Janisiw E., Rappol T., Vilardo E, & Schaefer M.R. (2023). Identification of RNA helicases with unwinding activity on angiogenin-processed tRNAs. Nucleic Acids Research, 51(3), 1326-1352.

+ Open protocol

+ Expand

UV-crosslink RNA Immunoprecipitation Assay

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

UV-crosslink RNA immunoprecipitation assay was performed as previously described with some modifications (Mineo et al., 2016 (link)). Briefly, cells were UV irradiated at 400 mJ/cm2 and nuclear extracts were prepared by incubating cells in RLN Buffer (50 mM Tris, 1.5 mM MgCl2, 150 mM NaCl, 0.5% NP-40, protease inhibitors) for 5 min. Nuclei were pelleted by centrifuging at 1,450 × g for 2 min and lysed for 10 min in CLIP Buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% Sodium Deoxycholate, phosphatase and protease inhibitors, 100 U/ml RNase inhibitor [New England BioLabs]). Samples were sonicated with microtip, 5 watts power (25% duty) for 60 seconds total in pulses of 1 second on followed by 3 seconds off. DNA was digested incubating samples for 15 min at 37°C in 1X DNase salt solution (2.5 mM MgCl2, 0.5 mM CaCl2) with 30 U TurboDNase. EDTA was added to the samples to a final concentration of 4 mM and samples centrifuged at 16,000 × g for 10 min. Nuclear extracts were precleared with Protein A/G Plus Agarose beads (Thermo Fisher Scientific) and incubated with primary antibody (anti-hnRNP-H) or rabbit IgG control (Bethyl Laboratories) overnight at 4°C. Protein/RNA complexes were precipitated using Protein A/G Plus Agarose beads (Thermo Fisher Scientific). Beads were washed and incubated with Proteinase K (Thermo Fisher Scientific) and RNA was extracted using TRIzol.

Mineo M., Lyons S.M., Zdioruk M., von Spreckelsen N., Ferrer-Luna R., Ito H., Alayo Q.A., Kharel P., Larsen A.G., Fan W.Y., Auduong S., Grauwet K., Passaro C., Khalsa J.K., Shah K., Reardon D.A., Ligon K.L., Beroukhim R., Nakashima H., Ivanov P., Anderson P.J., Lawler S.E, & Chiocca E.A. (2020). Tumor interferon signaling is regulated by a lncRNA INCR1 transcribed from the PD-L1 locus. Molecular cell, 78(6), 1207-1223.e8.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!