Gairdia parasites were iced for 30 min and pelleted at 700 x g for 7 min. The pellet was fixed in PME buffer (100 mM Piperazine-N,N’-bis (ethanesulfonic acid) (PIPES) pH 7.0, 5 mM EGTA, 10 mM MgSO4 supplemented with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), 100 µM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 µM ethylene glycol bis (succinimidyl succinate) (Pierce), and 0.025% Triton X-100 for 30 min at 37°C. Fixed cells were attached on polylysine coated coverslips. Cells were washed once in PME and permeabilized with 0.1% Triton X-100 in PME for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was incubated for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus 4’,6-diamidino-2-phenylinodole (DAPI; Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4-numerical aperture objective with a PCO Edge sCMOS camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
3 maleimidobenzoic acid n hydroxysuccinimide ester
Manufactured by Merck Group
3-maleimidobenzoic acid N-hydroxysuccinimide ester is a chemical compound used in various laboratory applications. It functions as a crosslinking agent, capable of forming covalent bonds between biomolecules such as proteins and other compounds.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Fish skin gelatin, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions)
Prolong gold antifade plus 4 6 diamidino 2 phenylinodole dapi, by Thermo Fisher Scientific (3 mentions)
Sepharose 6b, by Cytiva (1 mentions)
Cold water fish skin gelatin, by Merck Group (1 mentions)
Ethylene glycol bis succinimidyl succinate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
7 protocols using 3 maleimidobenzoic acid n hydroxysuccinimide ester
1
Immunofluorescence Staining of Giardia Parasites
2
Purification and characterization of antibodies
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Anti-BteA, anti-BopD, anti-Bsp22, BspR, BopB, and anti-Bcr4 antibodies were purified from rabbit serum in our previous study (6 (link), 7 (link), 10 (link), 14 (link), 24 (link), 38 (link)). Mouse anti-V5 and anti-RNA polymerase beta subunit (RpoB) monoclonal antibodies were purchased from Santa Cruz Biotechnology and BioLegend, respectively. To detect filamentous hemagglutinin (FHA) signals, we used mouse anti-FHA serum kindly provided by K. Kamachi (National Institute of Infectious Diseases). To prepare the anti-BscI and anti-BscJ antibodies, the peptides corresponding to the C-terminal regions of BscI (CKAIGRATQNVDTLARMS ) and BscJ (CRGEGRGGAGAGATEGAGHD ) were conjugated with hemocyanin from keyhole limpets (Sigma), respectively, by using 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma). These cross-linked peptides were used to immunize rabbits, and the resulting antisera were incubated with the same peptides immobilized on epoxy-activated Sepharose 6B (Amersham) to obtain specific Ig fractions.
3
Immunofluorescence Localization of CWP1 in Giardia
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Gairdia parasites were iced for 30 min and pelleted at 700 x g for 7 min. The pellet was fixed in PME buffer (100 mM Piperazine-N,N’-bis (ethanesulfonic acid) (PIPES) pH 7.0, 5 mM EGTA, 10 mM MgSO4 supplemented with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), 100 μM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 μM ethylene glycol bis (succinimidyl succinate) (Pierce), and 0.025% Triton X-100 for 30 min at 37°C. Fixed cells were attached on polylysine coated coverslips. Cells were washed once in PME and permeabilized with 0.1% Triton X-100 in PME for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was incubated for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus 4’,6-diamidino-2-phenylinodole (DAPI; Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4-numerical aperture objective with a PCO Edge sCMOS camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
4
Immunofluorescence Imaging of Giardia Cyst Wall
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Giardia parasites were iced for 30 min and pelleted at 700 × g for 7 min. The pellet was fixed in PME buffer (100 mM piperazine-N,N′-bis [ethanesulfonic acid], pH 7.0; 5 mM EGTA; 10 mM MgSO4 supplemented with 1% paraformaldehyde [PFA]; Electron Microscopy Sciences, Hatfield, PA), 100 µM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 µM ethylene glycol bis (succinimidyl succinate; Pierce), and 0.025% Triton X-100 for 30 min at 37 °C. Fixed cells were attached to polylysine-coated coverslips. Cells were washed once in PME and permeabilized with PME plus 0.1% Triton X-100 for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold-water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647–conjugated anti-CWP1 antibody (Waterborne) was added to incubate for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus DAPI (Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.42–numerical aperture objective with a PCO Edge sCMOS (scientific Complementary Metal–Oxide–Semiconductor) camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
5
Giardia Cell Division Synchronization
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To increase the rate of cell division, Giardia cells grown for 72 h were incubated for 4 to 5 h with fresh medium, as previously described (62 (link)). The harvested trophozoites were fixed on a slide with PBS containing 2% paraformaldehyde, 100 μM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), and 100 μM ethylene glycol-bis(succinimidyl succinate) (Thermo Fisher Scientific) for 30 min at 37°C. The cells were then treated with 100 mM glycine in PBS for 5 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. After three washes with PBS, the slides were blocked with 3% BSA in PBS for 30 min, incubated with primary antibodies, and then treated with secondary antibodies. After staining, the slides were mounted and observed under a Zeiss LSM700 confocal microscope (Carl Zeiss).
The following antibodies were used at the indicated dilutions: anti-HA rat monoclonal antibodies (clone 3F10, Roche Applied Science; 1:100), anti-acetylated-α-tubulin mouse antibodies (clone 6-11B-1, T7451, Sigma-Aldrich; 1:800), anti-Myc mouse antibodies (Thermo Fisher Scientific; 1:50), anti-Glcentrin mouse antibodies (59 (link)), Alexa Fluor 555-conjugated donkey anti-rat IgG (Molecular Probes; 1:100), and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Molecular Probes; 1:100).
The following antibodies were used at the indicated dilutions: anti-HA rat monoclonal antibodies (clone 3F10, Roche Applied Science; 1:100), anti-acetylated-α-tubulin mouse antibodies (clone 6-11B-1, T7451, Sigma-Aldrich; 1:800), anti-Myc mouse antibodies (Thermo Fisher Scientific; 1:50), anti-Glcentrin mouse antibodies (59 (link)), Alexa Fluor 555-conjugated donkey anti-rat IgG (Molecular Probes; 1:100), and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Molecular Probes; 1:100).
6
Antibody Production and Purification
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Anti-BteA, anti-BopB, and anti-Bsp22 antibodies were purified from rabbit serum in our previous study (10, 18, 29) . To detect filamentous hemagglutinin (FHA) signals, we used mouse anti-FHA serum (30) . Mouse anti-CyaA monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To prepare the anti-BtrS antibody, the peptide corresponding to the C-terminus region of BtrS (CALREALRERGYDSVP) were conjugated with hemocyanin from keyhole limpets (Sigma) by using 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma).
These cross-linked peptides were used to immunize rabbits, and resulting anti-sera were incubated with peptide immobilized on epoxy-activated sepharose 6B (Amersham) to obtain specific Ig-fractions.
These cross-linked peptides were used to immunize rabbits, and resulting anti-sera were incubated with peptide immobilized on epoxy-activated sepharose 6B (Amersham) to obtain specific Ig-fractions.
7
Microscopy of Giardia Parasites
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Gairdia parasites were iced for 30 min and pelleted at 700 x g for 7 min. The pellet was fixed in PME buffer (100 mM Piperazine-N,N'-bis (ethanesulfonic acid) (PIPES) pH 7.0, 5 mM EGTA, 10 mM MgSO4 supplemented with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA), 100 µM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 µM ethylene glycol bis (succinimidyl succinate) (Pierce), and 0.025% Triton X-100 for 30 min at 37C. Fixed cells were attached on polylysine coated coverslips. Cells were washed once in PME and permeabilized with 0.1% Triton X-100 in PME for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN3, 100 mM lysine, 0.5% cold water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647-conjugated anti-CWP1 antibody (Waterborne, New Orleans, LA) was added to incubate for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus 4',6-diamidino-2-phenylinodole (DAPI; Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4numerical aperture objective with a PCO Edge sCMOS camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).
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