In tissue, samples of 15 μg were loaded on 4–12% Bis-Tris SDS-PAGE (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat dry milk in PBS-Tween 0.1% and incubated against MMP-9 (Millipore, ab19016) at a concentration of 1:1000 overnight at 4°C. GAPDH or β-tubulin was used as loading controls. Detection was carried out using peroxidase-coupled anti-rabbit (Vector) at 1:10000 for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL). Cell media was thawed to room temperature and mixed with 4x SDS at a 1:3 ratio. Samples of 15 μl were loaded on 4–12% Bis-Tris SDS-PAGE (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat dry milk in PBS-Tween 0.1% and incubated against MMP-9 (Millipore ab19016) at a concentration of 1:1000 overnight at 4°C. Cell count was used for normalization. Detection was carried out using peroxidase-coupled anti-rabbit (Vector) at 1:10000 for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL).
Ab19016
Manufactured by Merck Group
Sourced in United States
The AB19016 is a laboratory equipment product manufactured by Merck Group. It is a general-purpose instrument used for various scientific applications. The core function of this product is to perform controlled measurements and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bis tris sds page, by Thermo Fisher Scientific (1 mentions)
Peroxidase coupled anti rabbit, by Vector Laboratories (1 mentions)
Novolink polymer detection system, by Leica Microsystems (1 mentions)
Ncl ki67 mm1, by Leica Biosystems (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mabcam 9484, by Abcam (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Histone h3 1g1, by Santa Cruz Biotechnology (1 mentions)
5hh2 2a8, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Las 4000, by Cytiva (1 mentions)
Gelatin, by Bio-Rad (1 mentions)
Renaturation buffer, by Bio-Rad (1 mentions)
Development buffer, by Bio-Rad (1 mentions)
13 protocols using ab19016
1
Quantifying MMP-9 Expression
2
Prostate Cancer Tissue Microarray Analysis
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A prostate adenocarcinoma tissue microarray was obtained from IMGENEX (IMT-01254, San Diego, CA) and contained 95 tissue cores representing samples from 48 cases of prostate cancer and 15 matched normal adjacent tissues. Mice bone xenograft tissues were collected at the end of the animal experiments (5 weeks after initial treatment). IHC staining was performed using the Novolink Polymer Detection System (Leica Microsystems, Newcastle Upon Tyne, UK) as previously described [16 (link)]. Antibodies used in the study included mouse anti-human L1CAM (1:40; clone UJ127, NeoMarker), mouse anti-human ki-67 (1:50; NCL-Ki67-MM1, Leica Biosystems), mouse anti-MMP-2 (1:50; clone MMP2/8B4, LifeSpan Biosciences), rabbit anti-MMP-9 (1:100; AB19016, Millipore), and rabbit anti-human NF-κb (1:100; clone E379, Millipore). Scoring of immunostaining of L1CAM in tissue microarray was done by two pathologists from the Department of Pathology, Emory University School of Medicine (Atlanta, GA) using a semi-quantitative scoring method according to the staining intensity and area extent, which has been widely accepted and used in our previous studies [63 (link), 64 (link)]. L1CAM expression was identified as any identifiable cytoplasmic/membranous staining, and quantified in 25% increments by visual estimation. Intensity of staining was recorded as weak, moderate and strong.
3
Quantifying Brain Protein Levels by Western Blotting
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To measure levels of GA and MMP-9 proteins in each brain region, Western blotting was performed with purified specific polyclonal antibodies against KGA and GAB, as well as commercial antibodies against MMP-9 (Millipore, Spain AB19016, 1/1000). Protein samples (40 μg for KGA and GAB or 20 μg for MMP-9 detection) were separated on SDS-PAGE gels and transferred to nitrocellulose or PVDF membranes. After blocking at room temperature for 1 h with 5% bovine serum albumin (BSA) in TBST buffer (0.1% Tween 20 in Tris-buffered saline, TBS, 100 mM Tris, 150 mM NaCl, pH 7.5), membranes were incubated with primary antibodies in blocking buffer with 5% BSA, overnight at 4°C. After incubation with secondary antibody, the blots were developed by enhanced chemiluminescense technique as recommended by the supplier (Pierce) and bands quantified using the Chemi Doc System (BioRad). β-actin was quantified and used as a loading control.
4
Western Blotting of Cell Lysate Proteins
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Western blot was performed as described8 (link). Briefly, total cell lysates were prepared in 100–400 µl RIPA buffer containing Protease Inhibitor cocktail (PhosStop, Roche; Protease Inhibitor, Sigma). The blots were incubated with anti-Mmp-9 (1:750, rabbit anti-mouse, AB19016, Millipore), anti-cytokeratin 16 (1:500, rabbit anti-mouse, NB100-91842, Novus Biologicals), anti-involucrin (1:1000, rabbit anti-mouse, PRB-140C-200, Covance), anti-Vdac1 (1:250, sc-32063, Santa Cruz), anti-phospho-PKC (pan) (1:1000, 9371, Cell Signaling Technology), or anti-Gapdh (1:2000, mAbcam 9484, AbCam) primary antibodies and subsequently with fluorescent IRDye 800 (1:1000, goat anti-rabbit, 611-132-122, Rockland), IRDye 800 (1:1000, donkey anti-goat, 925–32214, LI-COR Biotechnology), Alexa Fluor 680 (1:1000, donkey anti-mouse, A10038, Life Technologies) or Cy 5.5 (1:1000, goat anti-mouse, 610-113-121, Rockland) secondary antibodies. Bands were visualized using the Odyssey imaging system (LI-COR Bioscience) according to the manufacturer’s protocol. The samples (n = 3–4) were loaded on one blot and analysed (each sample comprised cultured cells isolated from 6–8 newborn mice). Densitometric protein analysis for Vdac1 was performed with ImageJ software, available at http://rsb.info.nih.gov/ij/index.html .
5
Western Blot Analysis of Histones
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The samples were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was incubated for 1 h with Blocking One (Nacalai Tesque) to block nonspecific binding. The membrane was then incubated overnight at 4 °C with antibodies against histone H2B (5HH2-2A8, Millipore), histone H1.2 (19649-1-AP, Proteintech), histone H2A (938CT5.1.1, Santa Cruz), histone H3 (1G1, Santa Cruz), histone H4 (L64C1, Cell signaling), RAGE (A-9, Santa Cruz), and MMP-9 (AB19016, Millipore) in 10% Blocking One-PBST, followed by the incubation for 1 h at room temperature with HRP-conjugated secondary antibodies (1:5,000 dilution, anti-mouse IgG, Cell signaling, #7076; 1:5,000 dilution, anti-rabbit IgG, Cell signaling, #7074). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and LAS4000 (Cytiva). The data were analyzed by ImageJ Fiji software version 1.52n. For the detection of total proteins, PVDF membrane was stained with 0.1% Ponceau S solution containing 5% acetate for 5 min. The stained membrane was scanned with LAS4000, destained with PBST, and then used for subsequent blocking and immunodetection for histone H2B.
6
Zymography and Western Blot Analysis of MMP-9
7
Quantifying Aortic MMP-2 and MMP-9 Localization
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In order to assess MMP-2 and MMP-9 localization in the thoracic aorta, the de-waxed aortic sections were analyzed using specific antibodies (ab37150; Abcam and ab19016; Millipore, respectively), and an immunohistochemical technique using the R.T.U. Vectastain ABC kit (Vector Laboratories, Inc., CA, USA) as previously described [40] (link). The experimental conditions were optimized to generate a strong and specific signal for the antigen of interest. The measurements were performed in a single batch and the specimens were stained together with a positive control at the same time. The stained sections was examined with light microscopy and the images were captured at ×400. The amount of MMP2 or MMP-9 in the aortic wall was measured, as were collagen and elastin, by counting the thresholded pixels stained with antibodies to MMP-2 or MMP-9 using the Image-Pro Plus Program (Media Cybernetics, MD, USA). All measurements were made by one observer and preliminary observations of intra-observer repeatability showed a coefficient of variation of less than 5% for all estimations of thresholded area.
8
Immunohistochemical Analysis of Lung Tissue Biomarkers
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Expressions of ORMDL3, MMP-9, p-ERK, VEGF, and CD31 in lung tissues were assessed by immunohistochemical staining. Samples were incubated with antibodies directed to ORMDL3 (Abcam, 107639), p-ERK (CST 4370), MMP-9 (Millipore, AB19016), VEGF (Millipore, ABS82), and CD31 (Abcam, ab28364) overnight, respectively. Then, samples were detected with biotinylated secondary antibodies and SAB reagent (Beijing Chinese fir Jinqiao). Five fields of each specimen were reviewed, and samples were assessed using Image J software.
9
Lung Protein Expression Analysis
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Lung tissue protein was determined though a Bio-Rad protein assay, to which proteinase inhibitors were added. Forty microgram tissues of lung were loaded on to an SDS-PAGE with antibodies directed to ORMDL3 (molecular weight: 17 kDa Abcam107639, 1:1,000), ERK (molecular weight: 42 kDa, CST 9102, 1:1,000), p-ERK (molecular weight: 44 kDa, CST 4370, 1:1,000), MMP-9 (molecular weight: 92 kDa, Millipore AB19016, 1:1,000), and GAPDH (1:2,000).
10
Immunohistochemical Analysis of MMPs and E-Cadherin
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