Antibodies used for Western blotting were diluted in PBS + 2.5% milk. Primary rabbit antibody to full-length Xenopus H1M (Maresca et al., 2005 (link)) was used 1:1,500. Primary antibody to histone H2A (rabbit, ab13923; Abcam) was used 1:1,000. Nap1 antisera were used at 1:4,000. Primary antibody to β-tubulin (mouse anti–tubulin E7; Developmental Studies Hybridoma Bank) was used at 1:5,000. Primary antibodies for γ-glutamylation, called TTβIII-glu and TTSG1 (rabbit, created against short synthetic glutamylated peptides derived from sequences from rat brain tubulin) were used at 1:10,000 as described in Spano and Frankfurter (2010) (link). TTβIII-glu and TTSG1 antibodies were provided by D. Shechter (Albert Einstein University, Bronx, NY). Secondary antibodies from Rockland Immunochemicals (goat anti–rabbit IRDye 800 or goat anti–mouse IRDye 700) were used at 1:10,000. Blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences), and band intensity was quantified with ImageJ.
Protocol detail
Quantitative Western Blot Analysis
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Antibodies
Antibody
Antisera
Brain
Goat
Histone h2a
Hybridoma
Irdye 800
Milk
Mouse
Nap1
Peptides
Rabbit
Tubulin
Xenopus
Corresponding Organization :
Other organizations : University of California, Berkeley
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Variable analysis
independent variables
- Dilution of primary antibodies in PBS + 2.5% milk
- Western blot band intensity
- Antibody targets (Xenopus H1M, histone H2A, β-tubulin, γ-glutamylation)
- Secondary antibodies (goat anti-rabbit IRDye 800, goat anti-mouse IRDye 700)
- Imaging system (Odyssey Infrared Imaging System)
- Quantification method (ImageJ)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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