GAPDH (6c5), E-cadherin (H-108), Vimentin (V9) and MMP-2 (H-76) antibodies were purchased from Santa Cruz Biotechnology (PasoRobles, CA). Phospho-Akt (Ser473 D9E) and Akt (11E7) antibodies were purchased from Cell Signaling Technology Company (Boston, MA), Phospho-mTOR (ab109268) and mTOR (ab32028) antibodies were from Abcam Company (San Diego, CA). CD68 (M087601-2) antibody were purchased from DAKO (Carpinteria, CA). Rapamycin was from Cell Signaling Technology Company (Boston, MA). Crystal violet was from Fishers Scientific Company (Grand Island, NY). Anti mouse/rabbit second antibody for Western Blot and Lipofectamine 2000 transfection reagent were purchased from Life Technologies Company (Grand Island, NY).
Mmp 2 h 76
Manufactured by Santa Cruz Biotechnology
Sourced in United States
MMP-2 (H-76) is a laboratory reagent produced by Santa Cruz Biotechnology. It is an antibody that specifically recognizes the Matrix Metalloproteinase-2 (MMP-2) protein. MMP-2 is an enzyme involved in the breakdown of extracellular matrix components.
Automatically generated - may contain errors
Lab products found in correlation
Western blotting luminol reagent, by Santa Cruz Biotechnology (2 mentions)
α sma 1a4, by Merck Group (2 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Hybond, by Cytiva (2 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Anti mouse rabbit second antibody for western blot, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Mtor ab32028, by Abcam (1 mentions)
Phospho akt ser473 d9e, by Cell Signaling Technology (1 mentions)
Akt 11e7, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc system instrument, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Ist 9, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer 10, by Cell Signaling Technology (1 mentions)
Timp 1, by Santa Cruz Biotechnology (1 mentions)
Hybond nitrocellulose membrane, by GE Healthcare (1 mentions)
4 protocols using mmp 2 h 76
1
Protein Expression Analysis Protocol
2
Protein Expression and Gelatin Zymography
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Cells were lysed in 10 mmol/L trisaminomethane hydrochloride buffer pH 7.4 with
2% sodium dodecyl sulphate (SDS) and fresh protease inhibitors. Protein
concentrations were determined by colorimetric assay after extracts sonication
for 20 s (Pierce™ BCA Protein Assay Kit, Thermo Scientific, Waltham,
Massachusetts, USA). The following primary antibodies were used to perform
western blotting: Bcl2L10 (#3869, Cell Signaling, Danvers, MA, USA),
p44/42 [extracellular-signal-regulated kinase (ERK)1/2, #9102,
Cell Signaling], phosphorylated p44/42 (ERK1/2, #9106 L, Cell
Signaling) and MMP2 (H-76, sc-10736, Santa Cruz Biotechnology, Santa Cruz, CA).
β-actin (#A1978, Sigma-Aldrich) and heat shock protein
(HSP)72/73 (#HSP01, Calbiochem, San Diego, CA, USA) were used to check
equivalent transfer and loading. Chemiluminescent method (Pierce, Rockford, IL)
was used to detect immunostained bands. Image Lab™ Software (Bio-Rad,
Hercules, CA, USA) and ChemiDoc System instrument (Bio-Rad), were used to
acquire images, while ImageJ software was used for densitometric evaluation and
normalization with relative controls.
Cultured medium (CM) from M14-B and M14-C cells incubated in serum free medium
(SFM) for 24 h was normalized to the number of adherent cells and assayed for
gelatinase activity using 7.5% SDS gels containing gelatin (0.1 mg/mL)
as previously described [31 (link),
32 (link)].
2% sodium dodecyl sulphate (SDS) and fresh protease inhibitors. Protein
concentrations were determined by colorimetric assay after extracts sonication
for 20 s (Pierce™ BCA Protein Assay Kit, Thermo Scientific, Waltham,
Massachusetts, USA). The following primary antibodies were used to perform
western blotting: Bcl2L10 (#3869, Cell Signaling, Danvers, MA, USA),
p44/42 [extracellular-signal-regulated kinase (ERK)1/2, #9102,
Cell Signaling], phosphorylated p44/42 (ERK1/2, #9106 L, Cell
Signaling) and MMP2 (H-76, sc-10736, Santa Cruz Biotechnology, Santa Cruz, CA).
β-actin (#A1978, Sigma-Aldrich) and heat shock protein
(HSP)72/73 (#HSP01, Calbiochem, San Diego, CA, USA) were used to check
equivalent transfer and loading. Chemiluminescent method (Pierce, Rockford, IL)
was used to detect immunostained bands. Image Lab™ Software (Bio-Rad,
Hercules, CA, USA) and ChemiDoc System instrument (Bio-Rad), were used to
acquire images, while ImageJ software was used for densitometric evaluation and
normalization with relative controls.
Cultured medium (CM) from M14-B and M14-C cells incubated in serum free medium
(SFM) for 24 h was normalized to the number of adherent cells and assayed for
gelatinase activity using 7.5% SDS gels containing gelatin (0.1 mg/mL)
as previously described [31 (link),
32 (link)].
3
Protein Extraction and Western Blot Analysis
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The cells were harvested, suspended in PBS containing 4 mM EDTA and washed 3 times. Collagen gels were dissolved. The proteins were extracted with Cell lysis buffer 10× (Cell Signaling Technology, Danvers, USA). Protein content was determined by Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich). Forty µg of protein were applied on Novex NuPAGE 4-12% Bis-Tris gel (Invitrogen Life Technologies, Prague, Czechia) under nonreducing conditions. The proteins were transferred to 0.2 mm Hybond nitrocellulose membrane (GE Healthcare, München, Germany). Staining with Ponceau S was used as a loading control. The membranes were incubated with antibodies to α-SMA (1A4, Sigma-Aldrich), cellular fibronectin (IST-9, Santa Cruz Biotechnology, Santa Cruz, USA) or MMP-2 (H-76, Santa Cruz), TIMP-1 (Santa Cruz) at 4 °C overnight. The secondary antibodies were from Santa Cruz. Detection was done with Western Blotting Luminol Reagent (Santa Cruz). The blots were scanned, quantified by program QUANTITY ONE 4.6 (Bio-Rad Laboratories, Hercules, California) and normalized to the respective controls.
4
Protein Analysis of Cell Cultures
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The cells were scraped from the plastic, suspended in PBS containing 4 mM EDTA and washed 3fold with this solution. Collagen gel was dissolved by Clostridium histolyticum collagenase NB 4G proved grade (Serva, Heidelberg, Germany). The cells were washed 3fold with EDTA-containing PBS. The proteins were extracted with cell lysis buffer (Cell Signaling). Protein content was determined with bicinchoninic acid (Sigma). Ten µg protein were applied on Novex NuPAGE 4-12 % Bis-Tris gel (Invitrogen Life Technologies, Prague) under nonreducing conditions. The proteins were transferred to 0.2 µm Hybond nitrocellulose membrane (GE Healthcare, München, Germany). Staining with Ponceau S was used as a loading control. The membranes were incubated with antibodies to α-SMA (1A4, Sigma), cellular fibronectin (IST-9; Santa Cruz Biotechnology, Heildelberg, Germany) or MMP-2 (H-76, Santa Cruz), TGF-β1 (V, Santa Cruz), Smad2/3 (D7G7, Cell Signaling), Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, Cell Signaling) at 4 °C overnight. The secondary antibodies were from Santa Cruz. Detection was done with Western Blotting Luminol Reagent (Santa Cruz). The blots were scanned, quantified by program QUANTITY ONE 4.6 and normalized to the respective controls.
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