SERCA2a stable line cells were transfected with mCer-PS1 two days before experiments. Cells were collected and resuspended in lysis buffer as previously described [12 (link)]. After quantification of total protein concentration, equal amounts of total cell lysates were run on a 4–20% SDS-Page gel and blotted using the Turbo transfer system. PS1 was probed with a mouse monoclonal anti-PS1 antibody specific to the C-terminal fragment (Millipore, USA) at a dilution of 1:1000. This antibody recognizes both the full length PS1 and the endocleaved C-terminal fragment. Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP at a dilution of 1:5000 and a chemiluminescence detection system (Biorad, USA).
Goat anti mouse secondary antibody
Manufactured by Bio-Rad
Sourced in United States
Goat anti-mouse secondary antibody is a laboratory reagent used in immunoassays and other immunological techniques. It is designed to bind to mouse primary antibodies, allowing for the detection and visualization of target analytes or antigens in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Visionworks ls analysis software, by Analytik Jena (2 mentions)
Goat anti rabbit secondary antibody, by Bio-Rad (2 mentions)
Anti mmp9, by Abcam (2 mentions)
Anti γh2ax, by Abcam (2 mentions)
Chemiluminescence detection system, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
Tmb peroxidase eia substrate, by Bio-Rad (1 mentions)
Donkey anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti hsp90, by BD (1 mentions)
Anti hsp90, by Cell Signaling Technology (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti c myc, by Abcam (1 mentions)
Anti claudin 4, by Thermo Fisher Scientific (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti claudin 2, by Thermo Fisher Scientific (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
11 protocols using goat anti mouse secondary antibody
1
Western Blot Analysis of PS1 Protein
2
Protein Extraction and Western Blotting
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Total protein was extracted from infiltrated systemic leaves as described previously (Li et al., 2014 (link)). Western blotting was performed with primary mouse monoclonal antibody [green fluorescent protein (GFP) or PVX CP protein monoclonal antibody, HuaAn Beijing, China], followed by goat anti-mouse secondary antibody conjugated to alkaline peroxidase (Bio-Rad). Blotted membranes were washed thoroughly and visualized using NBT/BCIP.
3
Quantifying Apoptosis-Related Proteins
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Total protein was extracted from splenic tissues using RIPA buffer (Sigma-Aldrich, USA) supplemented with protease inhibitor cocktail (Sigma Aldrich Co, LLC, USA). Samples of 20 μg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. Membranes were probed using specific primary antibodies against GAPDH (Abcam ab8245), Bcl-2 (Abcam ab59348), Bax (Abcam ab32503), or cytochrome-C (Abcam ab13575) at 4°C overnight. After washing 3 times (for 5 min each) in TBST, the ALP-conjugated goat anti-rabbit secondary antibody (Bio-Rad ab20571 for anti-Bcl-2 and anti-Bax primaries) or goat anti-mouse secondary antibody (Bio-Rad 170-6464 for anti-GAPDH anti-cytochrome-C primary) was applied, and membranes were then incubated with NBT-BCIP buffer for 15 min in the dark. Protein bands were visualized and captured using a Bio-Rad Gel Doc system and quantified by densitometry using molecular imaging software (ImageJ 148-JDK 6 software).
4
Quantitative ELISA for Viral NS1 Protein
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MDCK or A549 monolayers (overnight confluent, 48-well plate format, triplicates) were infected with assembled viruses at moi = 10. After virus adsorption at 37 °C, the inoculum was removed and replaced with serum free medium containing antibiotic-antimycotic and TPCK-trypsin. Infected cells were incubated at 37 °C for the indicated time periods and then fixed with 80% acetone in DPBS (ice cold at application) for 15 min at room temperature. Fixed cells were washed three times with phosphate buffered saline with 0.1% tween-20 (PBST) and blocked with 5% milk in PBST for 6 h at 4 °C. After 2 washes, plates were incubated at 4 °C overnight with mouse polyclonal serum raised against recombinant NS1(1–124) protein. Plates were then washed three times with PBST, and goat anti-mouse secondary antibody (Bio-Rad) was added (1 μg/ml final), followed by incubation at 37 °C for 1 h. Immunoreactivities were analyzed by adding TMB Peroxidase EIA Substrate (Bio-Rad), and reactions were stopped with 2 N H2SO4. Optical density was measured at 450 nm with removal of noise (measured at 655 nm) using a CLARIOstar multi-function reader (BMG Labtech).
5
Quantification of Myc Protein Levels
6
Evaluating Colonic Tight Junction Integrity
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As described previously [10 (link)], for WB analysis, colonic mucosal stripping was obtained from the TgM9 and WT mice (n=20 per group) with and without CAC. In vivo WB analysis was performed with 30μg/ well of cell lysates. WB analysis for in vitro model was performed using whole cell lysates (30μg/ well) of CaCo2BBE cells with and without MMP9. 106 cells of CaCo2BBE were grown on 6 well plate for 100% confluency. As described by Bazzoni et al [62 (link)], we collected Triton X-100 soluble and insoluble fraction of CaCo2BBE cells with and without MMP9 to assess TJ integrity and 10μg/ well was used for WB analysis. The antibodies used were anti-MMP9 (Abcam, Cambridge, MA), anti-EGFR (Cell Signaling, Beverly, MA), anti-Claudin-2 (Life Technologies, Rockford, IL), anti-Claudin-4 (Invitrogen, Rockford, IL), anti-Claudin-5 (Invitrogen), anti-TFF3 (Cloud-Clone Corp., Katy, TX), anti-Sp1 (Upstate Cell Signaling Solutions, Lake Placid, NY), anti-Occludin (Invitrogen), anti-STAT3 (Cell Signaling). Goat anti-mouse secondary antibody (Bio-Rad, Hercules, CA) or goat anti-rabbit secondary antibody (Bio-Rad) were used. Densitometry graphs were generated by using image acquisition and analysis software by VisionWorksLS Analysis Software (UVP, Upland, CA).
7
Western Blot Analysis of CHL1 in Mouse Colon
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The lysates were prepared from mouse distal colon tissues, and the total protein concentration was measured. Each sample was loaded onto an SDS-PAGE gel and was separated by electrophoresis. Then, the proteins were transferred to nitrocellulose membranes. After the membranes were blocked, the specific primary antibodies (CHL1, 1:1,000 dilution, R&D; β-actin, 1:10,000 dilution, Sigma) were applied overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated rabbit anti-goat (1:5,000 dilution; Bio-Rad, Hercules, CA) or goat anti-mouse secondary antibody (1:10,000 dilution; Bio-Rad) for 1 h at room temperature. The specific bands were visualized using an ECL detection kit (Bio-Rad).
8
Mucosal Protein Analysis in Colitis-Associated Cancer
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As described previously [10 (link)] mucosal stripping were obtained from the TgM9 and WT mice (n = 20 per group) with and without CAC after the sacrifice for WB analysis. The antibodies used were anti-MMP9 (Abcam, Cambridge, MA), anti-NICD (Abcam), anti-caspase-3 (Cell Signaling, Beverly, MA) anti-p53 (Cell Signaling), anti-p21WAF1/Cip1 (BD Bioscience, San Jose, CA), anti-cyclin D1 (Santa Cruz, Dallas, TX), anti-Cyclin A (Cell Signaling), anti-Cyclin E1 (Santa Cruz), anti-γH2AX (Abcam), anti-p19ARF (Abcam), and anti-p14ARF (Abcam). Goat anti-mouse secondary antibody (1:2000; Bio-Rad, Hercules, CA) or goat anti-rabbit secondary antibody (1:2000, Bio-Rad) were used. Densitometry graphs were generated by using image acquisition and analysis software by VisionWorksLS Analysis Software (UVP, Upland, CA)
9
Western Blot Analysis of SPA- and His-Tagged Proteins
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Gels were transferred to BioTrace
nitrocellulose membranes (VWR) at 30 V for 16 h or at 100 V for 1
h. Blots were blocked in 3% BSA for 1 h at room temperature on a shaker.
To probe for SPA-tagged proteins, 1:1000 dilution of mouse monoclonal
anti-FLAG M2 (Sigma) primary antibody was incubated with the blot
for 1 h, followed by washing with Tris buffered saline containing
0.1% Tween 20 (TBS-T). Goat anti-mouse secondary antibody (Rockland)
at a dilution of 1:10 000 was incubated for 1 h, followed by
washing with TBS-T. Blots were developed using Clarity ECL western
blotting substrate (Bio-Rad) and imaged using a ChemiDoc imaging system
(Bio-Rad) and Image Lab software (BioRad). For His6-tagged
proteins, His tag antibody conjugated to biotin was used. For detection,
streptavidin conjugated to AlexFluor 488 was incubated with the blot
for 30 min, followed by washing and analysis of the blot by a Typhoon
imaging system and Image Quant software (GE Life Sciences).
Changes in protein expression of SPA-tagged proteins were assessed
by quantifying the bands using Image Lab software (Bio-Rad). After
background subtraction, the fold change in expression was calculated
by dividing the intensity of bands at 10 °C by those at 37 °C.
At least three biological replicates were carried out for each protein
as well as the wild-type E. coli K12
MG1655 control.
nitrocellulose membranes (VWR) at 30 V for 16 h or at 100 V for 1
h. Blots were blocked in 3% BSA for 1 h at room temperature on a shaker.
To probe for SPA-tagged proteins, 1:1000 dilution of mouse monoclonal
anti-FLAG M2 (Sigma) primary antibody was incubated with the blot
for 1 h, followed by washing with Tris buffered saline containing
0.1% Tween 20 (TBS-T). Goat anti-mouse secondary antibody (Rockland)
at a dilution of 1:10 000 was incubated for 1 h, followed by
washing with TBS-T. Blots were developed using Clarity ECL western
blotting substrate (Bio-Rad) and imaged using a ChemiDoc imaging system
(Bio-Rad) and Image Lab software (BioRad). For His6-tagged
proteins, His tag antibody conjugated to biotin was used. For detection,
streptavidin conjugated to AlexFluor 488 was incubated with the blot
for 30 min, followed by washing and analysis of the blot by a Typhoon
imaging system and Image Quant software (GE Life Sciences).
Changes in protein expression of SPA-tagged proteins were assessed
by quantifying the bands using Image Lab software (Bio-Rad). After
background subtraction, the fold change in expression was calculated
by dividing the intensity of bands at 10 °C by those at 37 °C.
At least three biological replicates were carried out for each protein
as well as the wild-type E. coli K12
MG1655 control.
10
Colon Cancer Biomarker Analysis Protocol
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As described previously11 (link). WB analysis was performed by using the whole-cell lysates (30 μg per well) of the colonic mucosal stripping of TgM9 and their WT littermates’ mice with and without CAC. The primary antibodies used were anti-MMP9 (Cell Signaling, Beverly, MA, USA), anti-γH2AX (Abcam), anti-MDC1 (Novus Biologicals, Littleton, CO), anti-MLH1 (Abcam), anti-MSH2 (Abcam), and anti-PCNA (Abcam). Goat anti-mouse secondary antibody (Bio-Rad, Hercules, CA) or goat anti-rabbit secondary antibody (Abcam) were used. Anti-GAPDH (Abcam) or anti-β actin (Sigma) antibodies were used as the WB loading controls. Bio-Rad Quantity One software was used for densitometry analysis.
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