Protease and phosphate inhibitors

Manufactured by Thermo Fisher Scientific

Sourced in United States

Protease and phosphate inhibitors are laboratory reagents used to prevent the degradation of proteins and the dephosphorylation of phosphorylated molecules during sample preparation and analysis. They are designed to inactivate a wide range of proteases and phosphatases, ensuring the preservation of the native state of the target analytes.

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Lab products found in correlation

Pxi gel imaging system, by Syngene (2 mentions) Anti bmp2 monoclonal detection antibody conjugated to horseradish peroxidase, by Sino Biological (2 mentions) Anti ha antibody, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Xp rabbit mab, by Cell Signaling Technology (1 mentions) Mini protean, by Bio-Rad (1 mentions) Ecl detection system, by Bio-Rad (1 mentions) M per protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Nupage, by Thermo Fisher Scientific (1 mentions) Smad1 d59d7 xp rabbit mab, by Cell Signaling Technology (1 mentions) Superblock tbs blocking buffer, by Thermo Fisher Scientific (1 mentions) Ecl detection system, by Thermo Fisher Scientific (1 mentions)

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Protease inhibitors (1135 citations)

6 protocols using protease and phosphate inhibitors

Western Blot Analysis of BMP2 Signaling

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Cells were harvested following each transfection and lysed with 1X RIPA buffer, supplemented with protease and phosphate inhibitors (Thermo Fisher Scientific, Waltham, MA, Cat # 89900, A3278428, and A32953, respectively). Lysates (25–40 μg) were separated on Mini-PROTEAN™ (Bio-Rad, Hercules, CA) and transferred on to nitrocellulose membranes. The membranes were blocked for 1 hour with 5% milk in 0.05% TBST and probed with the following antibodies at 4°C overnight: anti-BMP2 monoclonal detection antibody conjugated to horseradish-peroxidase (Sino-Biological, Beijing, China, Cat No SEK 10426, dilution - 1:5000 – 1:10000), anti-HA antibody (Thermo Fisher Scientific, Cat # 14-6756-63, dilution 1:1000), Phospho-Smad1/5/9 (D5B10) Rabbit mAb (Cell Signaling, Danvers, CA, Cat No 13820T, dilution 1:500), Smad1 (D59D7) XP® Rabbit mAb (Cell Signaling, Danvers, CA, Cat No 13820T, dilution 1:1000). Blots were developed using the corresponding horseradish peroxidase-conjugated secondary antibodies with Immobilon™ chemiluminescence HRP substrate (Millipore, Billerica, MA, Cat # WBKLS0500) and the images were obtained with a PXi gel imaging system (Syngene, Frederick, MD, USA). Densitometric analysis was carried out using NIH-ImageJ™. Protein expression levels of all experimental conditions were represented as relative values (means ± SEM) of controls.

Khattar V., Lee J.H., Wang H., Bastola S, & Ponnazhagan S. (2019). Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion. FASEB bioAdvances, 1(3), 180-190.

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Western Blot Analysis of JAK-STAT Pathway

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BV-2 cells and tissues were lysed in RIPA buffer, containing protease and phosphate inhibitors (Thermo Scientific, Rockford, IL, USA). Protein concentrations in the cell and tissue homogenates were determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal quantities (40 μg) of proteins were separated on a 10% Sodium Dodecyl Sulfate (SDS)-polyacrylamide gel, transferred onto Polyvinylidene Fluoride (PVDF) membranes and blocked with 5% non-fat milk in TBST for 1 h at room temperature. The protein abundance was detected after incubating the primary antibodies against STAT3, phospho-STAT3, JAK2, phospho-JAK2, followed by HRP-conjugated secondary antibody. The blots were visualized by an enhanced chemiluminescence (ECL) detection system (Bio-Rad, Hercules, CA, USA).

Mao L., Hochstetter D., Yao L., Zhao Y., Zhou J., Wang Y, & Xu P. (2019). Green Tea Polyphenol (−)-Epigallocatechin Gallate (EGCG) Attenuates Neuroinflammation in Palmitic Acid-Stimulated BV-2 Microglia and High-Fat Diet-Induced Obese Mice. International Journal of Molecular Sciences, 20(20), 5081.

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Western Blotting Protein Quantification

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The complete cell lysates were harvested on ice with M-PER protein extraction reagent (Thermo Scientific, Waltham, MA, USA) containing a mixture of protease and phosphate inhibitors (Thermo Scientific, Waltham, MA, USA). After sonication for 10 seconds, cell debris was removed by centrifugation (12,000g) for 10 minutes at 4°C. The protein concentration was determined by BCA protein assay reagent (Pierce, Rockford, IL, USA). A total of 50ng of protein per lane was supplemented with loading buffer (NuPage; Invitrogen, Carlsbad, CA, USA) and boiled for 10 minutes. Immunoblotting was performed as previously described [18 (link)]. The cell lysates were subjected to 8%-16% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were probed with target antibodies.

Wang Z., Xiao X., Ge R., Li J., Johnson C.W., Rassoulian C, & Olumi A.F. (2017). Metformin inhibits the proliferation of benign prostatic epithelial cells. PLoS ONE, 12(3), e0173335.

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Western Blot Analysis of BMP2 Signaling

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Cells were harvested following each transfection and lysed with 1X RIPA buffer, supplemented with protease and phosphate inhibitors (Thermo Fisher Scientific, Waltham, MA, Cat # 89900, A3278428, and A32953, respectively). Lysates (25‐40 μg) were separated on Mini‐PROTEAN^TM (Bio‐Rad) and transferred on to nitrocellulose membranes. The membranes were blocked for 1 hour with 5% milk in 0.05% TBST and probed with the following antibodies at 4°C overnight: anti‐BMP2 monoclonal detection antibody conjugated to horseradish‐peroxidase (Sino‐Biological, Cat No SEK 10426, dilution ‐ 1:5000 ‐ 1:10000), anti‐HA antibody (Thermo Fisher Scientific, Cat # 14‐6756‐63, dilution 1:1000), Phospho‐Smad1/5/9 (D5B10) Rabbit mAb (Cell Signaling, Danvers, CA, Cat No 13820T, dilution 1:500), Smad1 (D59D7) XP® Rabbit mAb (Cell Signaling, Danvers, CA, Cat No 13820T, dilution 1:1000). Blots were developed using the corresponding horseradish peroxidase‐conjugated secondary antibodies with Immobilon^TM chemiluminescence HRP substrate (Millipore, Billerica, MA, Cat # WBKLS0500) and the images were obtained with a PXi gel imaging system (Syngene, Frederick, MD). Densitometric analysis was carried out using NIH‐ImageJ^TM. Protein expression levels of all experimental conditions were represented as relative values (means ± SEM) of controls.

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Vemurafenib Response in Melanoma Cells

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SK-mel 24 and SK-mel 28 cells were plated on 100-mm² dishes (~1.2 × 10⁶ cells) and incubated overnight to ensure cell adherence. The cells were washed with PBS and treated with vemurafenib or 0.1% DMSO (vehicle) for 6, 24, 48 or 72 hours (n = 3). Cells were then washed with PBS, lysed in radioimmunoprecipitation assay buffer containing protease and phosphate inhibitors (Thermo Fisher Scientific), left on ice for 30 minutes then concentrated by centrifugation at 14 000 rpm at 4°C for 10 minutes. The concentration of protein lysates was quantified using the bicinchoninic acid assay. For Western blot analysis, 25 μL of 20 μg/μL cell lysate was mixed with 25 μL 2x Laemmli loading buffer. Separated proteins in the gels were electrophoretically transferred onto polyvinylidene fluoride membranes. After washing and blocking, p-MEK1/2, p-ERK1/2, ERK1/2 and b-actin antibodies (1 mg/mL; Cell Signaling Technology) diluted in SuperBlock (TBS) blocking buffer (Thermo Fisher Scientific) were added and incubated overnight at 4°C. The bound antibodies were detected by horseradish peroxidase-conjugated antigoat Ig secondary antibody (Cell Signaling Technology) followed by ECL detection system (Thermo Scientific) according to the manufacturer’s instructions. Densitometry analysis was performed using ImageJ software; data were normalized to actin and expressed relative to the control.

Lees H., Millan M., Ahamed F., Eskandari R., Granlund K.L., Jeong S, & Keshari K.R. (2020). Multi-sample measurement of hyperpolarized pyruvate-to-lactate flux in melanoma cells. NMR in biomedicine, 34(3), e4447.

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Western Blot Analysis of ALDH1L1 in Liver

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Total protein was prepared from flash-frozen liver tissue. Approximately 300 mg liver tissue was minced and homogenized in 1 mL of RIPA buffer with protease and phosphate inhibitors (1:100) (Thermo Scientific, Waltham, MA, USA). Proteins were resolved on SDS polyacrylamide gel electrophoresis in 4–15% gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA) in transfer buffer containing 10% methanol. Membranes were probed with primary ALDH1L1-specific in-house polyclonal antibody (1:10,000) in Tris-buffered saline with Tween- 20 containing 5% nonfat milk. Horseradish peroxidase-conjugated secondary antibodies were used at 1:5000 dilution, and the signal was assessed with Super Signal West Pico chemiluminescence substrate (Pierce, Rockford, IL, USA).

Krupenko N.I., Sharma J., Fogle H.M., Pediaditakis P., Strickland K.C., Du X., Helke K.L., Sumner S, & Krupenko S.A. (2021). Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis. Cancers, 13(13), 3219.

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