Murine pancreatic β cells, NIT-1 cells (American Type Cell Culture, Manassas, VA), were seeded in culture platelet and treated with normal (7 mM) or high (25 mM) glucose culture medium for 24 or 48 h, respectively. Culture media from each well were collected, and uPA levels were measured by Mouse uPA Total Antigen Assay ELISA kit (Molecular Innovations, Novi, MI). Thereafter, equal amounts of protein (30 μg) from each well, after harvesting cells, were separated by gel. The gel was electroblotted onto a nitrocellulose membrane, and then incubated with 1:1000 dilutions of anti-uPA antibody (Abcam, Cambridge, MA), at 4 °C overnight. After incubation in horseradish peroxidase-conjugated anti-rabbit antibody, the membrane-bound antibody detected was incubated with Western blot detection system and captured on X-ray film.
Anti upa antibody
Manufactured by Abcam
Sourced in United States
Anti-uPA antibody is a laboratory reagent used to detect and quantify the urokinase plasminogen activator (uPA) protein. uPA is an enzyme involved in the breakdown of the extracellular matrix, and its expression is often associated with cancer progression and metastasis. The anti-uPA antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA, to measure the levels of uPA in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
200 μl cell lysis buffer, by Cell Signaling Technology (2 mentions)
β actin antibody, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti rabbit hrp conjugated secondary antibody, by BD (2 mentions)
Anti mouse hrp conjugated secondary antibody, by BD (2 mentions)
Mouse upa total antigen assay elisa kit, by Innovative Research (1 mentions)
Pd 10 size exclusion column, by GE Healthcare (1 mentions)
Secondary hrp antibody, by Jackson ImmunoResearch (1 mentions)
Chelex 100 resin, by Merck Group (1 mentions)
P scn bn df, by Macrocyclics (1 mentions)
Protein g agarose bead, by Thermo Fisher Scientific (1 mentions)
Ip lysis buffer, by Beyotime (1 mentions)
Protein g agarose beads, by Abcam (1 mentions)
6 protocols using anti upa antibody
1
Quantifying uPA Levels in Murine Pancreatic β Cells
2
Detailed Antibody Labeling and Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATN-291 was manufactured by SDIX [12 (link)]. Fluorescein isothiocyanate (FITC)- and Cy3-labeled secondary antibodies used in flow cytometry or histology were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, CA). Anti-uPA antibody, anti-uPAR antibody and anti-β-actin antibody (conjugated with horseradish peroxidase [HRP]) used in Western blotting were both purchased from Abcam (Cambridge, MA, USA). Secondary HRP antibodies were purchased from Jackson ImmunoResearch (St. Louis, MO, USA). p-SCN-Bn-Df (i.e. p-isothiocyanatobenzyl-desferrioxamine B) was acquired from Macrocyclics, Inc. (Cat #: B-705, Dallas, TX). Chelex 100 resin (50 – 100 mesh) was purchased from Sigma-Aldrich (St. Louis, MO). Buffers used in this study were prepared from Millipore-grade water and pre-treated with Chelex 100 resin to ensure that the aqueous solution was free of heavy metals. Size exclusion PD-10 columns were purchased from GE Healthcare (Piscataway, NJ). All other chemicals were purchased from Thermo Fisher Scientific (Fair Lawn, NJ).
3
Western Blot Analysis of uPA
4
NIT-1 Cell Responsiveness to uPA and Antibody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIT-1 cells were incubated with normal (7 mM) or high glucose (25 mM), respectively. The first group of NIT-1 cells was treated with 0.01 IU/mL or 1 IU/mL of uPA (Yao Chih Hsiang Inc., Taiwan) in culture medium. The second group of NIT-1 cells was treated with 0.6 nM or 60 nM of anti-uPA antibody (Abcam, Cambridge, MA, USA) in culture medium. The third group of NIT-1 cells was treated with uPA and uPA antibody at the same time. Insulin secretion rate and cell proliferation of β cells were measured (method as described above).
5
Immunoprecipitation of Urokinase-Type Plasminogen Activator
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IP was conducted with protein G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells after transfection were lysed and incubated in IP lysis buffer (Beyotime) for 10 min at room temperature. The extracts were incubated with anti-uPA-antibody (2 μg/ml, Abcam, Cambridge, UK) at 4 °C overnight, and the immunoprecipitants were purified by protein G agarose beads with gentle rocking. The beads were washed for three times with extraction buffer and resuspended in 20 μl SDS-loading buffer. The whole cell lysates and immunoprecipitants were incubated at 70 °C for 10 min followed with western blot analysis.
6
Western Blot Analysis of uPA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in 200 μl cell lysis buffer (Cell Signaling Technology; Danvers, MA) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland); protein concentrations were determined with the BCA protein assay (Thermo-Fisher, Waltham, MA). After electrophoresis on 4–12% SDS-PAGE gels, proteins were transferred to a PVDF membrane and blocked for 1 h in Tris-buffered saline with 5% nonfat milk at room temperature, similarly to what was previously described (Guizzetti et al., 2007 (link)). The PVDF membranes were incubated with an anti-uPA antibody (Abcam, Cambridge, UK; dilution 1:1000) for 2 h at room temperature followed by an anti-rabbit HRP-conjugated secondary antibody (BD Biosciences, San Jose, CA; dilution 1:5000) for 1 h at room temperature. After detection of uPA immunoreactivity, the membranes were stripped and re-probed with a β-actin antibody (Sigma, St. Louis, MO; 1:10,000 dilution) for 30 min followed by an anti-mouse HRP-conjugated secondary antibody (BD Biosciences, San Jose, CA; dilution 1:3000) for 30 min at room temperature. Densitometric analyses of uPA and β-actin immunoreactivity bands were carried out using the software ImageJ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!