谷胱甘肽（glutathione，r-glutamyl cysteingl +glycine，GSH）是一種含γ-酰胺鍵和巰基的三肽，由谷氨酸、半胱氨酸及甘氨酸組成，存在于幾乎身體的每一個細胞。胱甘肽有還原型（GSH）和氧化型（GSSG）兩種形式，在生理條件下以還原型谷胱甘肽占絕大多數。谷胱甘肽還原酶可以催化兩型間的互變。Virogen的谷胱甘肽抗體，貨號101-A，可以檢測谷胱甘肽化蛋白質復合物，當WB檢測時，需要在非還原條件下進行。具體WB檢測的數據，可以參考這篇文獻。
Antibodies
Glutathionylation was detected with the anti-glutathione antibody MAB5310 (Millipore). The immunoprecipitation of the glutathionylated proteins was carried out with a monoclonal anti-glutathioine (GSH) antibody (#101-A; Virogen). The Na,K-ATPase α1 isoform was immunoprecipitated with SC-21712 antibody (Santa Cruz Biotech., Dallas, TX) and immunedetected with the sc-28800 antibody (H-300; Santa Cruz Biotech.). All α subunits (α(all)) were immunoprecipitated with sc-28800 and immunodetected with the α5 antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City).The β1 isoform was immunodetected with a polyclonal antibody generously provided by Dr. P.A Pedersen, University of Copenhagen. The β2 isoform was detected with the polyclonal antibody 06-1711 (Millipore).
Quantification of glutathionylation
Western blotting of homogenized muscle material has shown that many proteins are susceptible to glutathionylation (Mollica et al. 2012). To study the glutathionylation of Na,K-ATPase subunits it is therefore necessary to use a purification step to isolate the subunits. The level of glutathionylation was studied with two independent techniques.
Method 1
Immunoprecipitation with Na,K-ATPase α and β subunit antibodies. The immunoprecipitate was divided in two parts and used in Western blots both for quantification of α and β subunits with other antibodies and for quantification of glutathionylation with the anti-GSH antibody (MAB5310) (and a sample buffer without the reducing agent dithiothreitol, DTT). The (relative) glutathionylation was calculated as the ratio between the labeling with anti-GSH and the α and β subunit isoform labeling. The glutathionylation of α and β subunit in the homogenate could not be measured with this method due to the presence of other glutathionylated proteins, the yield of glutathionylated α and β subunit proteins could therefore not be calculated.
Method 2
Glutathionylated proteins were immunoprecipitated with the anti-GSH antibody (#101-A; Virogen, Watertown, MA) and afterwards samples were subjected to Western blotting and Na,K-ATPase isoforms were detected with α and β subunit antibodies (sample buffer including DTT). The glutathionylation of α and β subunits was evaluated by calculating the ratio between isoform labeling after immunoblotting (with anti GSH) and the total labeling in the homogenate.
Virogen貨號101-A谷胱甘肽抗體WB檢測文獻數據參考,WB數據圖片：
The level of α and β subunits glutathionylation, the effects of exercise and terbutaline. The glutathionylated proteins were isolated with immunoprecipitation using anti-glutathione antibodies and Na,K-ATPase subunits quantified with Western blots of the immunoprecipitate (Method 2). (A) H; examples of the β1 subunits detected in the Western blots of two different homogenates (10 μg protein in each lane). IP: β1 subunits detected with anti-β1 antibodies in immunoprecipitates (from100 μg protein) obtained from the same two homogenates using the anti-GSH antibody.
The relative level of glutathionylated Na,K-ATPase α subunits (Method 1). (A) H; Western blot of muscle homogenate (10 μg protein per lane) labeled with the anti-GSH antibody. IP left: Western blots of two different immunoprecipitates (from 200 μg proteins) using the anti-α(all) antibody (H-300) and labeled on the gel with the anti-α(all) antibody α5. IP right: Western blots of the same two immunoprecipitates labeled with the anti-GSH antibody.
