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MO-C40009A, Transforming Growth Factor Beta (TGF-β), anti-human

Affinity Purified, Mouse

IgG1, TB21

Review #1: by Wojciech Ornatowski

Submitted July 13, 2004

Use: Western Blot on lung epithelial cells.

Comments: MO-C40009A antibody worked very well in my hands. The bands yielded 25 kDa protein product. The procedure used was as follow: Lung epithelial cells were extracted directly into lysis buffer (10 mM Tris-HCl [pH 7.6], 5 mM EDTA, 50 mM NaCl, 5 ug/ml aprotinin, 1 ug/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40 [Sigma]). Equal amounts (30 ug) of protein were electrophoresed through 10% precasted gel from Bio-Rad. After being transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA), nonspecific binding was blocked with 5% nonfat milk. The blots were then incubated with the primary antibodies for detection iNOS (1:1,000). Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:10,000; Amersham, Arlington Heights, IL) was then used to detect the bound primary antibodies, and finally developed using enhanced chemiluminescence (ECL kit; Amersham). Photographs of the exposed and developed X-ray films were shown. For quantitative analysis, the films were subjected to densitometry by scanning the images and analyzed using 1D Image Analysis Software (Kodak Digital Science, New Haven, CT).
 

Dilution: Western blot 1:1,000 or Immunofluorescence 1:500

Revew #2: by Dimension Laboratories

Use: Immunostaining

Comments:

1. Slides were de-waxed, blocked with methanol-H₂0₂ and hydrated through alcohol to Tris-Buffered Saline (TBS).

2. Tissues were blocked with normal rabbit serum, and then incubated with various dilutions of primary antibody overnight at 4^oC.  The antibody was diluted in Antibody Stabilization Buffer manufactured by Dimension Laboratories.

3. Tissues were washed with TBS and incubated for 10 minutes with biotinylated anti-mouse Ig (Zymed Laboratories).  Following another wash, tissues were incubated for 5 minutes with Horseradish Peroxidase conjugated Streptavidin (Zymed Laboratories).  All of these incubations were performed at room temperature (22^oC).

4. Tissues were washed and color developed with AEC.

Section of breast carcinoma composed of cells with large irregular nuclei with prominent nucleoli, interspersed with small cells with densely stained nuclei.  Staining is positive in the areas of mixed cell populations, with several intensely stained large cells and possible syncitial formations.  There is less intense cytoplasmic staining in numerous large cells, and weak staining, possibly of serum TGF, in the background of positive areas of the tissue.  Substitution of the TGF antibody with non-specific mouse IgG gave no staining.  Magnification approximately 320X.

Increased magnification (approx. 650X) of the same area of tissue as in photograph (1).

Dilution: The antibody dilution was 1:1000. 

Related Categories: Monoclonal Antibodies to Cytokines