Total protein concentration in the plasma samples was determined using Pierce BCA Protein Assay (Thermo Fisher, Waltham, MA). General protein separation was carried out by electrophoresis on 4–20% gradient SDS-PAGE gels at 110 V for 1.25 h and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Transfer membranes were then incubated in blocking buffer (1% BSA in TBST) at 4 °C under gentle agitation overnight. Blocked membranes were probed with primary recombinant monoclonal antibodies for trypsin (ab200997, 1:1000, Abcam, Cambridge, MA), pancreatic lipase (ab124915,1:30000, Abcam), and Transferrin (ab82411, 1:10,000, Abcam) before incubated in the corresponding secondary antibody (ab205718, 1/5000, Abcam). Membranes were incubated with Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and then imaged via photo-sensitive autoradiography film (Genesee Scientific, San Diego, CA). Western blot images were digitally analyzed (gel analysis tool, Image J; NIH). The band density values for each of the western blots for trypsin and lipase were normalized to their respective transferrin values.
Ab200997
Manufactured by Abcam
Sourced in Canada
Ab200997 is a laboratory equipment product. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab183495, by Abcam (2 mentions)
Nb100 56576, by Novus Biologicals (2 mentions)
Pipa529118, by Thermo Fisher Scientific (2 mentions)
Ls c150813 1, by LSBio (2 mentions)
Ab205718, by Abcam (1 mentions)
Ab82411, by Abcam (1 mentions)
Pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab57222, by Abcam (1 mentions)
4 protocols using ab200997
1
Quantitative Western Blot Analysis
2
Immunohistochemical Analysis of COVID-19 Tissues
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4-μm-thick sections were cut from paraffin blocks of lung and liver tissues from COVID-19 patients and non-COVID-19 donors. IHC staining with an anti-FXa protein antibody (PIPA529118, Invitrogen), an anti-furin antibody (ab183495, Abcam), an anti-trypsin antibody (ab200997, Abcam), or an anti-plasmin antibody (LS-C150813–1, LSBio) as a primary antibody was performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Mouse tissues isolated from experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with anti-NP protein antibody (NB100–56576, Novus) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Mouse tissues isolated from experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with anti-NP protein antibody (NB100–56576, Novus) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
3
Histological Analysis of COVID-19 Tissues
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4-μm-thick sections were cut from paraffin blocks of lung and liver tissues from COVID-19 patients and non-COVID-19 donors. The following primary antibodies were used for IHC staining: an anti-FXa protein antibody (PIPA529118, Invitrogen, at a 1:500 dilution), an anti-furin antibody (ab183495, Abcam, at a 1:500 dilution), an anti-trypsin antibody (ab200997, Abcam, at a 1:500 dilution), or an anti-plasmin antibody (LS-C150813-1, LSBio, at a 1:500 dilution). IHC was performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Tissues isolated from the experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with an anti-NP protein antibody (NB100-56576, Novus, at a 1:500 dilution) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
Tissues isolated from the experimental mice were placed in 10% neutral buffered formalin for a minimum of 72 hours. After paraffin embedding, 4-μm-thick sections were cut from the blocks. H&E staining and IHC with an anti-NP protein antibody (NB100-56576, Novus, at a 1:500 dilution) as the primary antibody were performed by the Pathology Shared Resource Core at City of Hope Beckman Research Institute. Stained slides were mounted and scanned for observation.
4
Co-localization of Digestive Enzymes and Gut Hormones
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To determine whether ghrelin and/or nesfatin-1 co-localize with the digestive enzymes (sucrase-isomaltose; aminopeptidase A; trypsin and lipoprotein lipase), sections of intestine were incubated as described above with a mixture of primary antibody against ghrelin (mouse anti-rat ghrelin, Catalog # ab57222, Abcam, Toronto, ON, Canada) or nesfatin-1 (mouse anti-rat nesfatin-1, Catalog # ALX-804-854-C100, Enzo Life Sciences, Brockville, ON, Canada) and primary antibody against one of the four studied digestive enzymes (rabbit anti-human sucrase-isomaltase, Catalog # ab98872; rabbit antihuman aminopeptidase A, Catalog # ab109775; rabbit anti-human trypsin, Catalog # ab200997; rabbit antihuman lipoprotein lipase, Catalog # ab137821; all from Abcam, Toronto, ON, Canada). Each digestive enzyme antibody was diluted 1:200, at room temperature. Since heterologous antibodies were used in this work, positive immunostaining or immunoreactivity (IR) is referred to as sucrase-isomaltase-like IR, aminopeptidase A-like IR, trypsin-like IR and lipoprotein lipase-like IR. All these antibodies were previously used in goldfish (Bertucci et al., 2017b; Blanco et al., 2017b) . Western blot analysis using total proteins of pejerrey intestine was conducted to confirm the specificity of each antibody (Supporting Information Fig. S1). All antibodies show bands at the expected protein mass.
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