Proteinase inhibitor cocktail

Manufactured by Roche

Sourced in United States, Germany, Switzerland, China, Japan, United Kingdom

Proteinase inhibitor cocktail is a laboratory reagent used to prevent the degradation of proteins during sample preparation and analysis. It contains a mixture of chemicals that inhibit the activity of various types of proteases, which are enzymes that break down proteins. The core function of this product is to preserve the integrity of protein samples by minimizing proteolytic degradation.

Automatically generated - may contain errors

Lab products found in correlation

Pvdf membrane, by Merck Group (27 mentions) Phosphatase inhibitor cocktail, by Roche (18 mentions) Trizol reagent, by Thermo Fisher Scientific (17 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions) Ripa buffer, by Merck Group (12 mentions) Bca protein assay kit, by Thermo Fisher Scientific (9 mentions) β actin, by Merck Group (8 mentions) Ripa buffer, by Beyotime (8 mentions) Ripa lysis buffer, by Beyotime (8 mentions) Nitrocellulose membrane, by GE Healthcare (7 mentions) Hybond, by Cytiva (7 mentions) Bca protein assay, by Thermo Fisher Scientific (7 mentions) Ripa lysis buffer, by Merck Group (7 mentions) Bradford assay, by Bio-Rad (6 mentions) Ripa buffer, by Thermo Fisher Scientific (6 mentions) Phosphatase inhibitor cocktail, by Merck Group (6 mentions) Pvdf membrane, by Bio-Rad (6 mentions) Nitrocellulose membrane, by Bio-Rad (6 mentions) Ripa lysis buffer, by Solarbio (5 mentions) Gapdh, by Santa Cruz Biotechnology (5 mentions)

Spelling variants of this product

Proteinase inhibitors (217 citations) Proteinase inhibitors cocktail (20 citations) Cocktail of proteinase inhibitors (9 citations) Cocktail inhibitor (7 citations) Complete EDTA-free proteinase inhibitor cocktail tablet (5 citations) Proteinase inhibitors cocktail tablets (4 citations) Phosphatase and proteinase inhibitor cocktails (3 citations) Proteinase inhibitors cocktail (Complete, (3 citations) Inhibitor cocktails (2 citations) Proteinase cocktail inhibitors EDTA-free tablets (2 citations)

475 protocols using proteinase inhibitor cocktail

Protein-Protein Interaction Assays: Co-IP and GST-Pulldown

Cited 1 time

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

Co-immunoprecipitation assays and GST-pulldown assay were applied for detection of protein–protein interaction. Cells were lysed with 1 ml lysis buffer (containing 20 mM HEPES (pH 7.4), 1% Triton-100, 100 mM NaCl, 5 mM MgCl2, and EDTA-free proteinase inhibitor cocktail from Roche) for 1 h on ice. The cell lysates were clarified by spinning at 14,000 rpm for 30 min.
For co-immunoprecipitation assay, INS-1 cells lysates were immunoprecipitated with anti-Syt1 antibody. For GST-pulldown assay, 293T cells transfected with the indicated plasmids. The supernatants were incubated with GST-fusion protein coupled to GST-Sepharose 4B resin (GE Healthcare, cat. No. 45-000-139) at 4°C for 4 h. The proteins bound to GST sepharose beads were detected through western blot assay.
Western blot was performed as described [58 (link)]. Briefly, cells were lysed in RIPA buffer containing proteinase inhibitor cocktail (Roche, cat. no. 04693132001). The resulted cell lysates were separated by SDS-PAGE, the resolved proteins were transferred to PVDF membrane, and then the membrane was blocked with 5% milk in TBST, followed by incubation with primary antibodies and HRP-conjugated secondary antibody. The blots were detected using ECL system (Pierce, Rockford, IL, USA).

Zhuang R., Zhou Y., Wang Z., Cao Y., Chen J., Xu L., Ren Y., Zheng Y., Wei Z., Qiu H., Li L., Han Y., Yun Y., Chen X., Hong W, & Wang T. (2023). Rab26 restricts insulin secretion via sequestering Synaptotagmin-1. PLOS Biology, 21(6), e3002142.

+ Open protocol

+ Expand

Recombinant Protein Purification in E. coli

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

Constructs generated for recombinant protein expression were transformed into E. coli BL21 (DE3) (Life Technologies, USA). Protein expression was induced in cultures with OD600 of 0.6 (mid-log phase) using 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 28 °C for 4 h. Cells were collected by centrifugation and resuspended in 3 mL ice cold lysis buffer (50 mM Tris-HCl pH 8.0, 0.2 M NaCl, 10% (v/v) glycerol, 1% (v/v) Triton-X-100, 1× proteinase inhibitor cocktail (Roche, Mannheim, Germany). Resuspended cells were lysed twice on ice with 2 min sonication using a Bandelin SONOREX (Sigma-Aldrich, Sydney, Australia) sonication bath. Following cell lysis, cell pellets containing inclusion bodies were collected and 5 mL of inclusion body solubilisation buffer (50 mM Tris-HCl pH8.0, 0.2 M NaCl, 6 M guanidine-HCl, 1 mM DTT, 1× proteinase inhibitor cocktail (Roche, Germany) were added to the pellet and mixed at room temperature until the pellets solubilised. Affinity chromatography was used to enrich the His-tagged recombinant proteins using cOmplete Ni-NTA purification resin (Roche, Germany) in a 20 mL chromatography column according to manufacturer’s instructions.

Wu X., Bacic A., Johnson K.L, & Humphries J. (2020). The Role of Brachypodium distachyon Wall-Associated Kinases (WAKs) in Cell Expansion and Stress Responses. Cells, 9(11), 2478.

+ Open protocol

+ Expand

Nuclear Protein Extraction Protocol

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

The cells were harvested in PBS and centrifuged at 1,000 g for 10 min at 4°C. After carefully aspirating the supernatant, the cells were resuspended in 200 μL ice-cold BUFFER-I (10 mM Hepes (pH 8.0), 1.5 mM MgCl₂, 10 mM KCl, 1 mM dithiothreitol, and proteinase inhibitor cocktail (Roche Molecular Biochemicals)) and incubated for 15 min on ice to allow the cells to be lysed, followed by adding 20 μL IGEPAL-CA630. After vigorously vortexing for 10 s and centrifuging at 12,000 g for 5 min at 4 °C, the supernatant (cytoplasmic fraction) was carefully aspirated and the pellet was resuspended with ice-cold BUFFER-II (20 mM Hepes (pH 8.0), 1.5 mM MgCl₂, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol and proteinase inhibitor cocktail (Roche Molecular Biochemicals)) and vigorously vortexed. After vortexing, the suspension was placed on ice for 30 min before centrifuging at 15,000 g for 15 min at 4°C. The supernatant (nuclear extracts) was stored in aliquots at -80°C. These samples contained the nuclear proteins. Protein concentration in each sample was determined by the Lowry assay, and Western blot analysis was conducted in order to determine the protein expression.

Chen Y.F., Day C.H., Lee N.H., Chen Y.F., Yang J.J., Lin C.H., Chen R.J., Rajendran P., Viswanadha V.P, & Huang C.Y. (2017). Tanshinone IIA Inhibits β-Catenin Nuclear Translocation and IGF-2R Activation via Estrogen Receptors to Suppress Angiotensin II-Induced H9c2 Cardiomyoblast Cell Apoptosis. International Journal of Medical Sciences, 14(12), 1284-1291.

+ Open protocol

+ Expand

Immunoprecipitation and Mass Spectrometry of BicDR Complexes

Cited 1 time

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

Embryos (12–16 h old) were collected and lysed in homogenization buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM EDTA and 1 mM DTT and 1 tablet of proteinase inhibitor cocktail; Roche 11836170001). The aqueous phase of the lysate was collected after 1 h of centrifugation at 21,300 g at 4°C and centrifugation again for 25 min. Subsequently, one part of the aqueous phase was saved as input control, whereas the rest was incubated with Plus Sepharose G beads that were coated with anti-GFP antibody overnight at 4°C, following the protocol of Vazquez-Pianzola et al. (2017) (link). 5–7 washing steps with wash buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT and a half tablet of proteinase inhibitor cocktail; Roche 11836170001) were performed before the beads were either sent for mass spectrometry or prepared with the appropriate amount of SDS for SDS-PAGE and western blot analysis. SDS/PAGE bands that were present in the IP from the BicDR::GFP fusion protein were cut out of the gel and sent directly for mass spectrometry at the Proteomics and Mass Spectrometry Core Facility of the University of Bern. As a control, the equivalent regions of the control lanes were also cut out and used for a mass spectrometric analysis.

Jejina A., Ayala Y., Beuchle D., Höhener T., Dörig R.E., Vazquez-Pianzola P., Hernández G, & Suter B. (2024). Role of BicDR in bristle shaft construction and support of BicD functions. Journal of Cell Science, 137(2), jcs261408.

+ Open protocol

+ Expand

Immunoprecipitation and Mass Spectrometry of BicDR::GFP

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

12–16 hours old embryos were collected and lysed in homogenization buffer (25 mM Hepes pH 7.4; 150mM NaCl; 0.5mM EDTA pH 8.0; 1 mM DTT and 1 tablet of proteinase inhibitor cocktail; Roche 11836170001). The aqueous phase of the lysate was collected after 1 hour of centrifugation at 4°C and centrifuged again for 25 minutes. Subsequently, one part of the aqueous phase was saved as input control, while the rest was incubated with Plus Sepharose G beads that were coated with anti-GFP antibody overnight at 4°C. 5–7 washing steps with wash buffer (25 mM Hepes pH 7.4; 150mM NaCl; 0.5mM EDTA pH 8.0; 1 mM DTT and ½ tablet of proteinase inhibitor cocktail, Roche 11836170001) were performed before the beads were either sent for mass spectrometry or prepared with the appropriate amount of SDS for SDS/PAGE and Western blot. SDS/PAGE bands that were present in the IP from the BicDR::GFP-fusion protein were cut out of the gel and sent directly for mass spectrometry. As a control, the equivalent regions of the control lanes were also cut out and used for a mass spectrometric analysis.

Jejina A., Ayala Y., Hernández G, & Suter B. (2023). Role of BicDR in bristle shaft construction, tracheal development, and support of BicD functions. bioRxiv.

+ Open protocol

+ Expand

Purification of Recombinant Proteins from E. coli

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

Recombinant proteins expressed in E. coli BL21 cells were purified according to the manufacturer’s instructions. Cells expressing MBP or MBP-GW2 were resuspended and sonicated in a buffer containing 20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF, and proteinase inhibitor cocktail (Roche). Total protein extracts were loaded onto a column packed with amylose resin (New England Biolabs). Columns were washed with buffer containing 20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, and 1% Triton X-100, and MBP and MBP-GW2 recombinant proteins were eluted with 10 mM maltose.
To purify GST fusion proteins, E. coli cells expressing GST-CHT14, GST-GAPDH, and GST-PGK were resuspended and sonicated in buffer containing 50 mM Tris, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF, and proteinase inhibitor cocktail (Roche). Total protein extracts were loaded onto a column packed with glutathione resin (Pharmacia), and columns were washed with buffer containing 50 mM Tris, 50 mM NaCl, 1 mM EDTA, and 1% Triton X-100. The recombinant proteins GST-CHT14, GST-GAPDH, and GST-PGK were eluted using 10 mM glutathione. All protein concentrations were determined using the Bradford assay (Bio-Rad).

Lee K.H., Park S.W., Kim Y.J., Koo Y.J., Song J.T, & Seo H.S. (2018). Grain width 2 (GW2) and its interacting proteins regulate seed development in rice (Oryza sativa L.). Botanical Studies, 59, 23.

+ Open protocol

+ Expand

Quantitative Immunoblotting of Neural Proteins

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

Cultured cells were lysed in RIPA buffer (Thermo Fisher) with proteinase inhibitor cocktail (Roche). For brain tissues, mouse was perfused from heart with 20 mL of saline before dissection of the injured brain issue. The dissected brain tissue was lysed in T-Per buffer (Thermo Fisher) with proteinase inhibitor cocktail (Roche). The lysate was gently sonicated using probe sonicator (Sonic Ultracell) on ice, followed by centrifugation at 10 000 rpm (5 min at 4 °C). The supernatant was retained and protein concentration was measured by a BCA Protein Assay Kit (Pierce) using BSA as the standard. The precasted 4–12% Bis-Tris minigel (Invitrogen) was used. The following primary antibodies were used to blot the transferred protein on PVDF membranes: βIII tubulin (1:1000, cat# ab18207, Abcam); Synapsin (1:1000, cat# 2312, Cell Signaling); β-actin (1:5000, cat# 8456, Cell Signaling). The blotting signals were captured from an Amersham Imager 600 (GE Healthcare) device, and semiquantified using ImageJ (NIH).

Wang Z., Wang Y., Wang Z., Zhao J., Gutkind J.S., Srivatsan A., Zhang G., Liao H.S., Fu X., Jin A., Tong X., Niu G, & Chen X. (2015). Polymeric Nanovehicle Regulated Spatiotemporal Real-Time Imaging of the Differentiation Dynamics of Transplanted Neural Stem Cells after Traumatic Brain Injury. ACS nano, 9(7), 6683-6695.

+ Open protocol

+ Expand

Immunoprecipitation of AGO1 in Arabidopsis

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

9-day-old Arabidopsis roots of EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic lines in WT and ktn1-20 backgrounds were collected and ground into powder in liquid nitrogen. Cells were lysed in immunoprecipitation buffer (50 mM Tris 7.5, 150 mM NaCl, 4mM MgCl 2 , 2mM DTT, 10% Glycerol, 0.1% NP-40, 1x proteinase inhibitor cocktail (Roche)) by gentle rotation at 4°C for 30 min. Cell debris was removed by centrifugation twice at 15000 rpm for 20 min at 4°C. The supernatant was incubated with GFP-Trap_MA (ChromoTek) for 2 h with gentle rotation. The beads were washed 5 times with washing buffer (50 mM Tris 7.5, 150 mM NaCl, 4mM MgCl 2 , 2mM DTT, 10% Glycerol, 0.5% NP-40, 1x proteinase inhibitor cocktail (Roche). Beads were magnetically collected and 10 % GFP-AGO1 immunoprecipitates were boiled in 1X SDS sample loading buffer for protein gel blot analysis and the remainder was subjected to RNA extraction using the TRI reagent.

Fan L., Zhang C., Zhang Y., Stewart E.L., Jeż J., Nakajima K., & Chen X. (2021). Microtubules Promote the Non-cell Autonomy of MicroRNAs by Inhibiting their Cytoplasmic Loading into ARGONAUTE1 inArabidopsis.

+ Open protocol

+ Expand

Recombinant Protein Purification Protocol

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

The recombinant proteins were purified according to the manufacturer’s instructions. MBP and MBP–GW2 were purified by resuspending and sonicating the recombinant cells expressing these proteins in a buffer containing 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF, and a proteinase inhibitor cocktail (Roche, Basel, Switzerland). Total protein extracts were loaded onto a column packed with amylose resin (New England Biolabs). After washing the column with buffer containing 20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA and 1% Triton X-100, the recombinant proteins were eluted with 10 mM maltose. GST–EXPLA1, GST–EXPLA1m1, and GST–EXPLA1m2 were purified by resuspending and sonicating the cells expressing these proteins in buffer containing 50 mM Tris, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF, and a proteinase inhibitor cocktail (Roche). Total protein extracts were loaded onto a column packed with glutathione resin (Pharmacia, Piscataway. NJ, USA). After washing the column with buffer containing 50 mM Tris, 50 mM NaCl, 1 mM EDTA and 1% Triton X-100, the recombinant proteins were eluted with 10 mM glutathione. The protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA).

Choi B.S., Kim Y.J., Markkandan K., Koo Y.J., Song J.T, & Seo H.S. (2018). GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1. International Journal of Molecular Sciences, 19(7), 1904.

+ Open protocol

+ Expand

Immunoprecipitation of GFP-AGO1 from Arabidopsis Roots

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

9-day-old Arabidopsis roots of EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic lines in WT and ktn1-20 backgrounds were collected and ground into powder in liquid nitrogen. Cells were lysed in immunoprecipitation buffer (50 mM Tris 7.5, 150 mM NaCl, 4 mM MgCl₂, 2 mM DTT, 10% Glycerol, 0.1% NP-40, 1x proteinase inhibitor cocktail (Roche)) by gentle rotation at 4°C for 30 min. Cell debris was removed by centrifugation twice at 15000 rpm for 20 min at 4 °C. The supernatant was incubated with GFP-Trap_MA (ChromoTek) for 2 h with gentle rotation. The beads were washed 5 times with washing buffer (50 mM Tris 7.5, 150 mM NaCl, 4 mM MgCl₂, 2 mM DTT, 10% Glycerol, 0.5% NP-40, 1x proteinase inhibitor cocktail (Roche). Beads were magnetically collected and 10% GFP-AGO1 immunoprecipitates were boiled in 1X SDS sample loading buffer for protein gel blot analysis and the remainder was subjected to RNA extraction using the TRI reagent.

Fan L., Zhang C., Gao B., Zhang Y., Stewart E., Jez J., Nakajima K, & Chen X. (2022). Microtubules promote the non-cell autonomous action of microRNAs by inhibiting their cytoplasmic loading onto ARGONAUTE1 in Arabidopsis. Developmental cell, 57(8), 995-1008.e5.

+ 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!