Proteasearrest protease inhibitors, by G Biosciences - Product details

1

Isolation and Subtilisin Digestion of Axonemes

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

Axonemes from wild-type and mutant strains were isolated following a published protocol (Craige et al., 2013 ). Briefly, cells were deflagellated with 25 mM dibucaine (Sigma-Aldrich) in HMDS buffer (10 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 4% Sucrose, pH 7.4) and centrifugated at 1,800 × g for 5 min to remove cell bodies. The supernatant containing flagella was collected and laid over HMDS buffer containing 25% sucrose. After centrifugation at 2,400 × g for 10 min, the supernatant containing flagella was collected down to the sucrose interface. To remove membranes and matrix from flagella, NP-40 (USB Chemicals) was added to flagella to a final concentration of 1% and centrifuged at 30,000 × g for 20 min. The white pellet at the bottom of tube (representing the axonemes) was resuspended with HMDEKP buffer (30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, PH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences). In the last step, axonemes (equal to 32 OD₂₈₀) were mildly digested with 10 μg/ml subtilisin A (Sigma-Aldrich) on ice for 30 min in the presence of 2 mM ATP in a total reaction volume of 10 μl before plunge freezing. ProteaseArrest protease inhibitors were present in the buffer to minimize the effects of subtilisin digestion.

Ma M., Stoyanova M., Rademacher G., Dutcher S.K., Brown A, & Zhang R. (2019). Structure of the decorated ciliary doublet microtubule. Cell, 179(4), 909-922.e12.

+ Open protocol

+ Expand

2

Isolation and Characterization of B. malayi Proteins

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

B. malayi worms were procured from the Filariasis Research Reagent Resource Center (FR3, filariasiscenter.org, Athens, GA). To produce whole worm lysates, worms were subjected to one freeze-thaw cycle, incubated overnight at 4 °C in RIPA buffer with ProteaseArrest protease inhibitors (G-biosciences, St. Louis, MO), mechanically disrupted by pestle and sheared with a 25G needle, then clarified by centrifugation (13500 × g for 30 min). Thirty adult female, 30 adult male, and 10⁶ Mf were used for the respective whole worm lysates.
B. malayi ES products were collected from microfilaria or 10-week-old adult worms. Fifty-four female, 89 male, and 3×10⁶ Mf were respectively maintained in 10 or 20 mL RPMI 1640 media supplemented with L-glutamine and penicillin/streptomycin (Gibco) for 48 hours at 37 °C/ 5% CO₂. Spent media was concentrated and buffer exchanged into PBS by centrifugal filtration using Amicon Ultra 3000 MWCO filters (Millipore) to a final volume of 500 μl. Proteins were precipitated with trichloroacetic acid (20% final concentration) and incubated at −20 °C for one hour then centrifuged for 30 minutes at 13500 × g. The protein pellet was washed three times with cold acetone, air-dried, and resuspended in 20mM Tris-HcL pH 8.0.

Hertz M.I., Glaessner P.M., Rush A, & Budge P.J. (2019). Brugia malayi galectin 2 is a tandem-repeat type galectin capable of binding mammalian polysaccharides. Molecular and biochemical parasitology, 235, 111233.

+ Open protocol

+ Expand

3

Isolation and Dissociation of Chlamydomonas Axonemes

Cited 1 time

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

Axonemes were isolated from wild type and fap236 mutant Chlamydomonas cells and dissociated into microtubules as previously described^{2 (link)}. Briefly, cells in HMDS buffer (10 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 4% Sucrose, pH 7.4) were treated with 25 mM dibucaine (Sigma-Aldrich) to induce deflagellation. Cell bodies were removed by centrifugation at 1,800 x g for 5 min. The flagella-containing supernatant was collected and laid over a 25% sucrose suspension in HMDS buffer. After centrifugation at 2,400 x g for 10 min, the supernatant was collected down to the sucrose interface. NP-40 (USB Chemicals) was added to the flagella to a final concentration of 1% to remove membranes. Axonemes were collected by centrifugation at 30,000 x g for 20 min and then resuspended in HMDEKP buffer (30 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, pH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences). The volume and optical density (at 280 nm) of the purified axonemes was adjusted to 10 μL and 32 absorbance units. Then the sample was mildly digested in HMDEKP buffer containing 10 μg/mL subtilisin A (Sigma-Aldrich) and 2 mM ATP, in the presence of ProteaseArrest protease inhibitors. Protease digestion was performed on ice and stopped after 30 min by plunge freezing.

Gui M., Wang X., Dutcher S.K., Brown A, & Zhang R. (2022). Ciliary central apparatus structure reveals mechanisms of microtubule patterning. Nature structural & molecular biology, 29(5), 483-492.

+ Open protocol

+ Expand

4

Purification of Tektin Filaments

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

Tektin filaments were purified following a published protocol (Pirner and Linck, 1994 (link)). Briefly, purified cilia were demembranized by adding CHAPS detergent to a final concentration of 2% and incubated at 4°C for 30 min, followed by a centrifugation at 6,000 × g for 10 min. The pellet was resuspended in tektin buffer (0.5% Sarkosyl, 50 mM Tris, pH 8, 1 mM EDTA, 1 mM DTT). Urea was added to the sample to a final concentration of 2 M. The sample was incubated at room temperature for 1 hour before diluting the urea concentration to 0.8 M. The sample was then subjected to ultracentrifugation using a SW50.1 rotor at 100,000 × g for 90 min at 16°C. The supernatant was discarded, and the pellet was resuspended in 20 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM DTT, 1 mM EGTA, 100 mM KCl containing 1× ProteaseArrest protease inhibitors (G-Biosciences). The sample was denatured with SDS-loading buffer and loaded onto a 4%–20% precast polyacrylamide gel (Bio-Rad). The gel was silver stained and the bands between 10 kDa to 40 kDa were cut and sent for mass-spectrometry analysis. Additionally, another gel was run for 3 min and stained with Coomassie blue. The bands containing the whole sample was cut and sent for mass-spectrometry analysis.

Gui M., Farley H., Anujan P., Anderson J.R., Maxwell D.W., Whitchurch J.B., Botsch J.J., Qiu T., Meleppattu S., Singh S.K., Zhang Q., Thompson J., Lucas J.S., Bingle C.D., Norris D.P., Roy S, & Brown A. (2021). De novo identification of mammalian ciliary motility proteins using cryo-EM. Cell, 184(23), 5791-5806.e19.

+ Open protocol

+ Expand

5

Cellular Lysate Preparation for SPCP Analysis

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

To prepare total cell lysates, IEC-6 cells were cultured to 40–50% confluence and then maintained in SFM. After 4 h, SFM containing SPCP (0, 12.5, 25, and 50 μg/mL) was added to the cells, which were incubated for 24 h. Cells were washed twice with PBS and lysed with radioimmunoprecipitation assay (RIPA) buffer containing Protease Arrest protease inhibitors (G-Biosciences, St. Louis, MO, USA) on ice. Cell lysates were collected on ice, centrifuged at 18,000× g for 10 min at 4 °C, and then the supernatant was analyzed using bicinchoninic acid (BCA) reagent for western blotting.

Jeong S.J., Choi J.W., Lee M.K., Choi Y.H, & Nam T.J. (2019). Spirulina Crude Protein Promotes the Migration and Proliferation in IEC-6 Cells by Activating EGFR/MAPK Signaling Pathway. Marine Drugs, 17(4), 205.

+ Open protocol

+ Expand

6

Heme Biosynthesis Pathway Profiling

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

Heart tissue was excised, rinsed in PBS, and perfused with Hanks buffered saline solution using a Langerdorff heart perfusion system. Approximately 10 mg of perfused tissue was homogenized in RIPA buffer in the presence of Protease Arrest protease inhibitors (G-Biosciences), centrifuged at 5000g for 15 minutes to remove debris, and protein concentration of the supernatant determined by bicinchoninic acid assay. Fifty to 100 μg of protein were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Invitrogen). The membranes were probed with antibodies against HO-1 (Abcam, Cambridge, MA), ALAS1 (Abcam), ALAS2 (Abcam, Novus Biologicals, Littleton, CO), and GAPDH (Santa Cruz, Dallas, TX). Horseradish peroxidase–conjugated donkey anti-rabbit and donkey anti-mouse were used as secondary antibodies (Santa Cruz) and visualized by Pierce SuperSignal Chemiluminescent substrates.

Sawicki K.T., Shang M., Wu R., Chang H.C., Khechaduri A., Sato T., Kamide C., Liu T., Naga Prasad S.V, & Ardehali H. (2015). Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 4(8), e002272.

+ Open protocol

+ Expand

7

Preparation of Brugia malayi Antigens and Excretory/Secretory Products

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

The Filariasis Research Reagent Resource Center (FR3, www.filariasiscenter.org, Athens, GA) provided adult female B. malayi worms recovered from jirds 8–10 weeks post-infection. To prepare B. malayi antigens (BmA) from whole worm lysate, thirty worms were subjected to one freeze-thaw cycle and then incubated overnight at 4°C in RIPA buffer with ProteaseArrest protease inhibitors (G-biosciences, St. Louis, MO). Samples underwent mechanical disruption by pestle and then by shearing with a 25G needle with subsequent centrifugation (13500 × g for 30 min). To collect excretory/secretory (ES) products, 60 worms were cultured for two weeks at 37°C/5% CO₂ in 20 ml of RPMI 1640 media supplemented with L-glutamine, glucose and 100 U/mL penicillin/ 0.1 mg/mL streptomycin/ 0.25 μg/mL amphotericin B (Sigma). The spent media was collected on alternating days over 14 days and stored at −20°C.The ES products were concentrated to 0.1 to 0.2 μg/mL by centrifugal filtration using an Amicon Ultra 3000 MWCO filter (Millipore). L. loa adult worms were a gift from Dr. Vida Dennis, Tulane University. L. loa soluble antigen was prepared by grinding adult worms in extraction buffer (10 mM Tris pH 8.3, 2% sodium deoxycholate, 1 mM PMSF, 1 mM EDTA, 1 mM EGTA, 25 μg/mL TLCK protease inhibitor, 15 μg/mL TPCK protease inhibitor) and collecting the soluble fraction.

Hertz M., Rush A., Nutman T.B., Weil G., Bennuru S, & Budge P. (2020). Characterization of glycan determinants that mediate recognition of the major Wuchereria bancrofti circulating antigen by diagnostic antibodies. Molecular and biochemical parasitology, 240, 111317.

+ Open protocol

+ Expand

8

Purification of Tektin Filaments

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

Tektin filaments were purified following a published protocol (Pirner and Linck, 1994 (link)). Briefly, purified cilia were demembranized by adding CHAPS detergent to a final concentration of 2% and incubated at 4°C for 30 min, followed by a centrifugation at 6,000 × g for 10 min. The pellet was resuspended in tektin buffer (0.5% Sarkosyl, 50 mM Tris, pH 8, 1 mM EDTA, 1 mM DTT). Urea was added to the sample to a final concentration of 2 M. The sample was incubated at room temperature for 1 hour before diluting the urea concentration to 0.8 M. The sample was then subjected to ultracentrifugation using a SW50.1 rotor at 100,000 × g for 90 min at 16°C. The supernatant was discarded, and the pellet was resuspended in 20 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM DTT, 1 mM EGTA, 100 mM KCl containing 1× ProteaseArrest protease inhibitors (G-Biosciences). The sample was denatured with SDS-loading buffer and loaded onto a 4%–20% precast polyacrylamide gel (Bio-Rad). The gel was silver stained and the bands between 10 kDa to 40 kDa were cut and sent for mass-spectrometry analysis. Additionally, another gel was run for 3 min and stained with Coomassie blue. The bands containing the whole sample was cut and sent for mass-spectrometry analysis.

Gui M., Farley H., Anujan P., Anderson J.R., Maxwell D.W., Whitchurch J.B., Botsch J.J., Qiu T., Meleppattu S., Singh S.K., Zhang Q., Thompson J., Lucas J.S., Bingle C.D., Norris D.P., Roy S, & Brown A. (2021). De novo identification of mammalian ciliary motility proteins using cryo-EM. Cell, 184(23), 5791-5806.e19.

+ Open protocol

+ Expand

Proteasearrest protease inhibitors

Lab products found in correlation

Close spelling variants of this product

8 protocols using proteasearrest protease inhibitors

Isolation and Subtilisin Digestion of Axonemes

Isolation and Characterization of B. malayi Proteins

Isolation and Dissociation of Chlamydomonas Axonemes

Purification of Tektin Filaments

Cellular Lysate Preparation for SPCP Analysis

Heme Biosynthesis Pathway Profiling

Preparation of Brugia malayi Antigens and Excretory/Secretory Products

Purification of Tektin Filaments

About PubCompare

Ready to get started?