A custom-made human tissue blot was received from ProSci (Poway) containing 15 μg of total cellular proteins from testis, ovary, spleen, skeletal muscle, small intestine, liver, skin, and brain. The staining protocol was performed as described previously [32 (link)] using guinea pig anti-SMC6 (GP anti-SMC6; 1:200; custom made), rabbit anti-SMC6 (1:1000; ab18039; Abcam), and rabbit anti-SMC5 (1:200; sc-134544; Santa Cruz) as primary antibodies. As secondary antibodies, goat anti-rabbit and donkey anti-guinea pig IRDye 800CW (1:15 000; LI-COR Biosciences) were used. As a loading control, a primary goat anti-GAPDH antibody (1:1000; ab9483; Abcam) was used. Image acquisition and quantification were done with the Odyssey Infrared Imaging System (LI-COR Biosciences).
Protocol detail
Tissue Blot Immunostaining of SMC6 and SMC5
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Anti antibody
Antibodies
Brain
Cellular
Donkey
Gapdh
Goat
Guinea pig
Human
Irdye 800cw
Liver
Ovary
Proteins
Rabbit
Skeletal muscle
Skin
Small intestine
Spleen
Testis
Tissue
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Variable analysis
independent variables
- Primary antibodies used: guinea pig anti-SMC6 (GP anti-SMC6; 1:200; custom made), rabbit anti-SMC6 (1:1000; ab18039; Abcam), and rabbit anti-SMC5 (1:200; sc-134544; Santa Cruz)
- Expression levels of SMC6 and SMC5 proteins in the tested tissues (testis, ovary, spleen, skeletal muscle, small intestine, liver, skin, and brain)
- Total cellular proteins (15 μg) from each tissue used for the tissue blot
- Goat anti-GAPDH antibody (1:1000; ab9483; Abcam) used as a loading control
- Positive control: None specified
- Negative control: None specified
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