CD19+ B cells were isolated from the spleens of B6 mice, treated with CBirTox and then washed, and subsequently cultured with B6.CBir1 Tg CD4+ T cells for 48 hours. CD4+ T cells were then removed from the culture using BD IMag CD4+ magnetic beads, and the B cells were washed 3 times before being lysed. Protein concentrations were determined using the Micro BCA Protein Assay Kit (Pierce). Equal amounts of protein were loaded onto a 10% Tris-HCl gel and separated by SDS-PAGE. Protein was then transferred to immobilon-P transfer membranes (Millipore) and detected with the following antibodies: p70S6K, pp70S6K (Thr421/Ser424) (Thr389), and HRP-linked rabbit or mouse anti-IgG (Cell Signaling). Densitometry was performed using an AlphaImager 2000 (Alpha Innotech).
Alphaimager 2000
Manufactured by Bio-Techne
Sourced in United States, United Kingdom
The AlphaImager 2000 is a compact and versatile imaging system designed for a wide range of imaging applications. It features a high-resolution CCD camera, a motorized zoom lens, and a UV or white light transilluminator. The AlphaImager 2000 is capable of capturing images of gels, blots, and other samples with high resolution and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescent detection kit, by Cytiva (2 mentions)
Ne per extraction kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sas software, by SAS Institute (2 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
3 30 diaminobenzidine, by Merck Group (1 mentions)
Semi dry transfer apparatus, by Bio-Rad (1 mentions)
Protease inhibitors cocktail, by Merck Group (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Vibra cell sonicator, by Sonics (1 mentions)
Phosphostop, by Roche (1 mentions)
Mmp 2, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
X omat bt film, by Kodak (1 mentions)
Total smad2 3, by Cell Signaling Technology (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
20 protocols using alphaimager 2000
1
Analysis of p70S6K Activation in B Cells
2
Duodenal Protein Expression Analysis
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Each duodenal mucosa sample was scraped with a glass slide and homogenized [in RIPA (radio-immunoprecipitation assay) buffer containing 150mM NaCl, 50mM Tris-HCl (pH 8.0), 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulfate), 1mM PMSF (phenylmethylsulfonyl fluoride), 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 1mM sodium orthovanadate] with a homogenizer on ice [6 ]. The homogenate was centrifuged at 12,000g for 20 minutes at 4°C. Protein concentrations were determined using the Lowry et al’s [22 (link)] method. Samples in sample buffer (v/v = 1/1) were heated at 95°C for 5 minutes and then loaded on a 10% gel. The proteins separated were then transferred to polyvinylidene fluoride membranes using a semidry transfer apparatus (Bio-Rad, Philadelphia, PA, USA). Each membrane was blocked with 5% nonfat milk for 2 hours and probed with DMT-1, FPN, or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Santa Cruz Bioscience, CA, USA) antibodies diluted in Tris-buffered saline. Cross-reactivity was observed with peroxidase-linked anti-IgG. Colorimetric protein detection used 3,3′-0-dia-minobenzidine (Sigma Chemical, St. Louis, MO, USA). Band intensities were measured with an AlphaImager 2000 (Alpha Innotech, San Leandro, CA, USA).
3
Immunofluorescence Analysis of Protein Localization
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All experiments were performed at least three times using different oocyte batches or cell cultures and values are presented as mean ± SD. Oneway ANOVA was performed on all Pf determinations. Molecular weights and densitometry analyses were performed using software from alpha-imager 2000 (Alpha Innotech Corp, San Leandro, CA). Immunofluorescence data was gathered by measuring the number of positive cell (non-permeabiliezed fixation) in a random field along its adjacent 8 fields.
4
Western Blot Analysis of Signaling Proteins
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Total cell lysates or nuclear extracts were prepared as previously described [3 (link)]. The cell lysates were separated in a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. The blot was subsequently incubated with 5% non-fat milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) for 1 h to block non-specific binding, and then overnight with polyclonal antibodies against three MAPKs (ERK 1/2, JNK 1/2, and p38), CREBBP, EP300, acetyl-Histone-H3, acetyl-Histone-H4 and β-actin with the specific antibodies for unphosphorylated or phosphorylated forms. The blots were then incubated with horseradish peroxidase goat anti-rabbit or anti-mouse IgG for 1 h. Signal was detected by using an enhanced chemiluminescence (ECL) commercial kit (Amersham Biosciences, Piscataway, NJ, USA). The relative photographic density was quantitated by scanning the photographic negatives on a gel documentation and analysis system (AlphaImager 2000, Alpha Innotech Corporation, and San Leandro, CA, USA).
5
Western Blot Analysis of Protein Expression
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Cell lysates were separated in a 10% or 15% polyacrylamide gel and transferred onto a PVDF membrane (Millipore Corporation, Milford, MA, USA) [19] (link). The blot was subsequently incubated with 5% non-fat milk in PBS for 1 h to block non-specific binding, and probed with a corresponding antibody against a specific protein for 37°C at 2 h or overnight at 4°C, and then with an appropriate peroxidase conjugated secondary antibody for 1 h. After the final washing, signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis (AlphaImager 2000, Alpha Innotech Corporation, San Lean 189 dro, CA, USA) [20] (link).
6
G418 Selection of Transfected Cells
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Two days following transfection with the indicated plasmids, G418 (500 μg/mL) was added to the culture medium, and at day 14, the cells were stained using gentian violet. Untransfected cells were treated similarly, and all died within 2 weeks of culture in the selection medium. Quantification of the results was done using AlphaImager 2000 (Alpha Innotech, USA).
7
Measuring MMP-2 Activity by Gelatin Zymography
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The matrix metalloproteinase-2 (MMP-2) activity was evaluated using gelatin zymography. The Clone-9 cells were incubated in DMEM medium or PAW DMEM medium for 24 h after plating 8 × 104 cells in 24-well plates for 16 h. The harvested culture medium containing 10 g of total protein was subjected to SDS-PAGE containing 0.1% gelatin. After electrophoresis, the gel was washed with a washing buffer (2.5% Triton X100) to remove SDS and then incubated at 37 °C in a reaction buffer containing 40 mM Tris–HCl, 10 mM CaCl2, and 0.01% NaN3. The proteolytic activity of MMP-2 was obtained by staining with Coomassie Brilliant Blue R-250. The intensity of unstained bands on the blue background was measured by spot density measurement using a densitometer (AlphaImager 2000, Alpha Innotech Corp., San Leandro, CA).
8
Western Blot Analysis of Pulmonary Arterial Proteins
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Protein was extracted from PASMCs and pulmonary arterial tissues with lysis buffer (RIPA buffer: PMSF = 6:1), and equal amounts of protein from each sample (30 µg) were separated by 12% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies overnight at 4 °C and horseradish peroxidase-coupled goat anti-rat or anti-rabbit secondary antibody. The chemiluminescence signals were detected with a chemiluminescent HPR substrate (Millipore Corporation, Billerica, USA). The densitometric analysis was conducted with Alpha Imager 2000 (Alpha Innotech Corporation, San Leandro, USA).
9
Protein Expression and Quantification
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Cells were harvested by either scraping or trypsinization, washed with PBS, and re-suspended in phosphate buffer (pH 7.8). Protease and phosphatase inhibitors were added to the preparations of all protein extracts (Protease inhibitors cocktail, Sigma and Phospho-Stop, Roche). Samples were sonicated on ice, with 5 bursts of 15 s, using a Vibra Cell Sonicator with cup attachment (Sonics and Materials Inc., Newtown, CT, USA). Protein concentrations were determined with a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by 12.5% SDS-PAGE, electro-transferred by semidry blotting onto a nitrocellulose membrane, and probed with antibodies to MnSOD, cyclin B1, and Complex I. Immunoreactive bands were detected by an enhanced chemiluminescence kit. The bands were visualized using X-ray film and imaged with computerized digital imaging system using AlphaImager 2000 software (Alpha Innotech, San Leandro, CA, USA). Bands were quantified with ImageJ software (ImageJ, National Institute of Health, Bethesda, MD, USA). The integrated density value was obtained by integrating the entire pixel values in the area of one band after correction for background. Actin protein levels were used for loading corrections.
10
Kaempferol Modulates Cell Signaling
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786-O cells were seeded onto 10-cm dishes at a density of 6×105 cells and treated with kaempferol at a concentration of 0-100 μM at 37℃ for 24 h. Cell lysates were separated in a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. The blot was then incubated with standard blocking buffer for 1 h to block nonspecific binding and subsequently incubated with polyclonal antibodies against extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), p-ERK1/2 (Thr185/Tyr187), Akt, p-Akt (Ser473) (Invitrogen Corporation, Carlsbad, CA), focal adhesion kinase (FAK) (Cell Singling Technology, Inc., Danvers, MA) and monoclonal antibodies against p-FAK (Tyr397), phosphatidylinositol 3-kinase (PI3K) (BD Biosciences, Fairleigh, NJ) and MMP-2 (millipore, CA). The signal was detected by enhanced chemiluminescence and quantified using a densitometer (AlphaImager 2000, Alpha Innotech Corporation, San Leandro, CA, USA).
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