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Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS, as well as to regulate redox-sensitive signaling pathways General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, while the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein needed for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissue and cells. In general, these assays require 24 to 48 hours to complete.
Reactive oxygen species (ROS) are produced in many aerobic cellular metabolic processes. They include, but are not limited to, species such as superoxide and hydrogen peroxide which react with various intracellular targets, including lipids, proteins, and DNA1. Although ROS are generated during normal aerobic metabolism, the biological effects of ROS on these intracellular targets are dependent on their concentration and increased levels of these species are present during oxidative stress. Increased levels of ROS are cytotoxic, while lower levels are necessary for the regulation of several key physiological mechanisms including cell differentiation2, apoptosis3, cell proliferation4 and regulation of redox-sensitive signal transduction pathways5. However, increased levels can also result in ROS-induced damage including cell death, mutations, chromosomal aberrations, and carcinogenesis1.
The intracellular concentration of ROS depends on the production and/or removal by the antioxidant system. Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS, as well as to regulate redox-sensitive signaling pathways. Three of the primary antioxidant enzymes contained in mammalian cells that are thought to be necessary for life in all oxygen metabolizing cells6 are superoxide dismutase (SOD), catalase, and a substrate specific peroxidase, glutathione peroxidase (GPx) (Fig. 1). The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen (O2), while the catalase and peroxidases convert hydrogen peroxide into water and in the case of catalase to oxygen and water. The net result is that two potentially harmful species, superoxide and hydrogen peroxide, are converted to water. SOD and catalase do not need co-factors to function, while GPx not only requires several co-factors and proteins but also has five isoenzymes. In the glutathione system, glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PD) do not act on ROS directly, but they enable the GPx to function7. There are three SOD enzymes that are highly compartmentalized. Manganese-containing superoxide dismutase (MnSOD) is localized in the mitochondria; copper- and zinc-containing superoxide dismutase (CuZnSOD) is located in the cytoplasm and nucleus and extracellular SOD (ECSOD0 is expressed extracellularly in some tissues. Other compartmentalized antioxidant enzymes include catalase, which is found in peroxisomes and cytoplasm, and GPx, which can be found in many sub-cellular compartments including the mitochondria and nucleus depending on the family member. Thus, the many forms of each of these enzymes reduces oxidative stress in the various parts of the cell. Thus, antioxidant proteins with similar enzymatic activity may have different effects after modulation due to different localizations within cells.
CuZnSOD comprises approximately 90% of total SOD activity in a eukaryotic cell7. Besides its primary distribution in the cytosol, a small fraction of this enzyme has been found in cellular organelles such as lysosomes, peroxisomes, and the nucleus8. Recently, there has been some evidence showing the presence of CuZnSOD (approximately 2%) in the intermembrane space of mitochondria9,10 and this localization was suggested to be important in providing further protection against ROS and in preventing superoxide radicals from leaking out of the mitochondria. Although ECSOD also utilizes copper and zinc as catalytic cofactors in a similar fashion as CuZnSOD, ECSOD is the only isoform of SOD that is expressed extracellularly and is distributed in the extracellular matrix of many tissues11,12. ECSOD is highly restricted to specific cell types and tissues such as lung, heart, kidney, plasma, lymph, ascites, and cerebrospinal fluid12. Unlike the other SODs, ECSOD has affinity for heparin sulfate proteoglycans located on cell surfaces and in extracellular matrix due to its heparin-binding domain13. The heparin-binding domain is important because it mediates the binding of ECSOD to cells. Also, ECSOD is a glycosylated high molecular weight homotetramer (155 kDa), while CuZnSOD is an unglycosylated homodimer (32 kDa). MnSOD (88 kDa) is found in the mitochondrial matrix and is inducible in eukaryotes following treatment with paraquat, irradiation, and hyperoxia suggesting that MnSOD induction is important for protection against oxidative stress14. In addition, MnSOD has been found to be decreased in many cancer cell types and increasing MnSOD levels reverses the in vitro and in vivo malignant phenotype of many cancers15,16,17.
Catalase converts hydrogen peroxide to water and oxygen. Catalase activity is largely located in subcellular organelles known as peroxisomes. Targeted delivery of catalase to the liver by galactosylation suppresses hepatic metastasis and decreases matrix metalloproteinase (MMP) activity, while a decrease in catalase correlates with carcinogen-initiated emergence of the malignant phenotype in mouse keratinocytes18. Catalase also attenuates both the basal and MnSOD-dependent expression of MMPs and collagen deposition19.
Cytosolic glutathione peroxidase (GPx, GPx1) is a selenoprotein, first described as an enzyme that protects hemoglobin from oxidative degradation in red blood cells20. As seen in Figure 1, GPx requires several secondary enzymes (glutathione reductase and glucose-6-phosphate dehydrogenase) and cofactors (reduced glutathione, NADPH, and glucose 6-phosphate) to function at high efficiency. As mentioned previously, there are five GPx isoenzymes21 with GPx1 considered a major enzyme responsible for removing H2O2. Overexpression of this enzyme protects cells against oxidative damage22,23, suppresses apoptosis induced by H2O224,25, and reverses the malignant phenotype in pancreatic cancer7.
In our laboratory, we have used in-gel activity assays to determine the activity of SODs, catalase, and GPx15,16,17,26. The most important parameter determining the biological impact of the antioxidant enzymes is activity. Because the expression of the antioxidant enzyme mRNA or protein does not necessarily result in an increase in activity27, enzymatic assays and native gels (Fig. 2 and and3)3) are utilized to measure the activity of the antioxidant enzymes. The activity assay requires 10-fold more protein than the gel assays but gives a quantitative result while the native gel requires less protein but results in a qualitiative result. Additionally a visual image is often a compelling way to present or address a scientific question, thus histological images of cells in culture or of cells (Fig. 4) within a tissue sample can help to better understand basal and abnormal expression of proteins of interest. Immunohistochemical analysis is an ideal method for determining cell specific antioxidant expression levels (Fig. 5)26, 28. Highly specific immunostains for both tissues and cells are available for SOD, GPx, and catalase. MnSOD staining will be present in the mitochondria, CuZnSOD throughout the cytoplasm, GPx in the mitochondria and nucleus, and catalase in the peroxisome. The immunohistochemical (Fig. 5), immunofluorescence and immunogold (Fig. 6) methodologies used to determine endogenous SOD, catalase, and GPx are standard in most histology laboratories. The antibodies used in these applications are all available commercially and can be used on fresh or fixed tissue or cells. Representative images of MCF 10A cells immunostained for MnSOD and CuZnSOD demonstrate robust, specific staining (Fig. 4). The drawback of immunostaining is that immunohistochemistry does not measure the activity of the antioxidant protein and as mentioned, there is a potential, especially during disease states, that the protein can be expressed but remain inactive. Antioxidants can be measured in tissue or cell lysates, however homogenates of tissues contain a mixture of cell types potentially diluting the levels of a given antioxidant in a specific cell type within the sample. Furthermore, the subcellular location of the proteins of interest cannot be determined.
These protocols are of interest to any investigator who is involved in oxidative stress as a mechanism of nearly any field of biomedical study including but not limited to cancer, cardiovascular disease, immunology, and aging. These protocols will be applicable to investigations that focus on the question of whether increased oxidant formation, due to an alteration in antioxidant enzymes, is the cause of human disease. Additionally, these protocols will be beneficial to investigators who wish to know whether alterations in antioxidant enzymes contribute to the disease or is it just an epiphenomenon. For example, our laboratory has demonstrated that there are decreased levels of MnSOD in pancreatic cancer15,16,28. This has been demonstrated by immunohistochemistry in human pancreatic cancers28 and in human pancreatic cancer cell lines15,16. Moreover, we have shown that stable17 and transient15,16 increases in the MnSOD gene resulting in increased immunoreactive protein and activity, reverses the malignant phenotype of cells. In other diseases, the increased levels of proteins with minor antioxidant activity may be a consequence of the disease29 and not contribute to the disease pathology30. Thus, applications utilizing the measurement of antioxidant in cells and tissue are highly diverse and span all disciplines including genetics, biochemistry, physiology, neuroscience, and molecular biology.
SOD activity can be measured by both activity assays and activity gels. In the biochemical method, xanthine-xanthine oxidase is used to generate O2•− and nitroblue tetrazolium (NBT) reduction is used as an indicator of O2•− production. SOD will compete with NBT for O2•−; the percent inhibition of NBT reduction is a measure of the amount of SOD present. Catalase is included to remove H2O2 produced by SOD. In our laboratory15,16,17 SOD activity is measured using a modification of a published method31,32. The specific activity of both enzymes is reported as units per mg protein33, per μg DNA34, or per cell. The order of the addition of the reagents into the assay solution is critical. Bovine serum albumin (BSA) is added to keep the solution from forming precipitates when bathocuproine disulfonic acid (BCS) is added. BCS (an electron chain-associated free radical production inhibitor) and diethylenetriaminepentaacetic acid (DETAPAC) are both added to inhibit iron-associated redox cycling and free radical production. The original concentration of sample protein should be around 20 μg μl-1. Various amounts of protein are added into tubes until maximum inhibition of NBT reduction (~80%) is obtained. The protein amounts regularly used in our lab are as follows: For human or animal tissues and cultured cells with high SOD activity: 2, 5, 10, 15, 25, 50, 100, 200, 300, 500 μg protein/tube. For cultured cells with low SOD activity: 5, 10, 25, 50, 100, 200, 500, 800, 1200, 1500 μg protein/tube. While the amount of SOD in total will give you a good idea of the overall superoxide detoxifying capacity of a cell or tissue, determination of both total SOD and MnSOD, and subsequently CuZnSOD, will be more informative as location and responsiveness of MnSOD and CuZnSOD differ. Moreover, while MnSOD is considered inducible, CuZnSOD in some instances can also be enhanced in some disease states or in response to certain treatments35. Thus, differential determination of SOD activity should be performed in most cases.
Additional methods that can be used to determine SOD activity are the cytochrome c assay or the potassium superoxide-based direct assay. Determination of SOD activity with the NBT-NCS methodology presented here36 is a more sensitive assay than using cytochrome c as the reductant, the addition of DETAPAC and BCS to the assay greatly reduces the homogenate-associated background activity signal while maintaining the pH of the assay thus ensuring SOD can function efficiently. An alternative potassium superoxide-based direct assay analysis of SOD11 is determined at a pH that alters the activity of MnSOD. Furthermore, an additional stop-flow kinetics assay cannot accurately determine SOD within cell and tissue homogenates37.
The SOD activity gel assay carried out in our laboratory15,16,17 is also based on the inhibition of the reduction of NBT by SOD originally described by Ornstein38 and by the method of Davis39. The principle of this assay is based on the ability of O2•− to interact with NBT reducing the yellow tetrazolium within the gel to a blue precipitate. Areas where SOD is active develop a clear area (achromatic bands) competing with NBT for the O2•−. Once run, the gels are stained for SOD activity by the method of Beauchamp and Fridovich40. CuZnSOD and MnSOD can be differentiated by the presence of sodium cyanide in the staining solution, which inhibits CuZnSOD (Fig. 2). Stained native activity gels will have a light to dark purple appearance with clear bands representing the area where SOD enzymes are present. The SOD activity gel assay uses 12% gels to allow for both visualization of MnSOD (88 KDa) and the smaller CuZnSOD (32KDa) (Box 1).
In Figure 2, a representative gel, in grayscale, shows distinct MnSOD bands (upper bands) and the broader CuZnSOD bands (lower band). The CuZnSOD band can also be present as multiple distinct bands. These bands will not appear if exposed to sodium cyanide. Sodium cyanide will readily bind the copper within the active site of CuZnSOD, inhibiting its activity and not allowing the enzyme to compete with NBT. This assay is also a good alternative to the spectrophotometric activity assay if limited sample is available; 100 – 250 μg protein for the gel assay verses a minimum of 2.5 mg per sample for the spectrophotometic SOD assay.
Catalase activity in our laboratory is measured by a spectrophotometric procedure measuring peroxide removal15,26. It is a direct assay with pseudo-first order kinetics29,30 and is measured by the method of Beers and Sizer41. The rate of peroxide removal by catalase is exponential. It is difficult to saturate catalase due to the large rate constants of compound I and II (1.7 × 10-7 M-1 s-1, 2.6 × 10-7 M-1 s-1, respectively). Catalase will begin to be inactivated by H2O2 at levels greater than 0.1 M, when compound I is converted to compound II or III. By the end of the assay, H2O2 is consumed and catalase is inactivated42. Catalase activity gels can also be used and will be green-blue in color with white broad bands where the enzyme is present (Box 2). Following separation of native protein, the catalase enzyme removes the peroxides from the area of the gel it occupies. Removal of peroxide does not allow for the potassium ferricyanide (a yellow substance) to be reduced to potassium ferrocyanide that reacts with ferric chloride to form a Prussian blue precipitate43. Catalase gels (8% gels) will have one band (220 KDa) that rarely saturates getting larger with increasing catalase activity 15,26,30. Both of the catalase assays should be preformed utilizing a positive control. The catalase positive control activity is defined in international unit equals (1 unit) as the amount of catalase necessary to decompose 1.0 μM of H2O2 per minute at pH 7.0 at 25 °C while H2O2 concentration falls from ≈ 10.3 mM to 9.2 mM. The concentration of H2O2 can be calculated from absorbance using the following expression: [H2O2 mM] = (Absorbance 240 nm × 1000) / 39.4 mol-1 cm-1; where 39.4 mol-1 cm-1 is the molar extinction coefficient for H2O2.
We measure GPx activity in our laboratory7 according to an established procedure using H2O2 as a substrate44. However, the assay can be carried out with cumene hydroperoxide or tert-butyl hydroperoxide as the substrate instead of H2O2 to measure total GPx45. Tert-butyl and cumene hydroperoxides are both hydroperoxides and both glutathione S-transferases (non-selenium containing peroxidase or selenium independent) and GPx (contains a selenium in the active site) can utilize hydroperoxides to determine total peroxidase activity. On the other hand, glutathione S-transferases will not detoxify H2O2. Thus, the assay using cumene hydroperoxide or tert-butyl hydroperoxide measures selenium-dependent GPx and activity from glutathione S-transferases (selenium-independent GPx)7,15. This assay is an indirect, coupled assay for glutathione peroxidase. This assay takes advantage of glutathione disulfide (GSSG) formed by the enzymatic action of GPx and is regenerated by excess glutathione reductase (GR) in the assay. The action of GR is monitored by following the disappearance of the co-substrate NADPH at 340 nm as seen in the reaction scheme below (Figure 7, gray box). This is a modification of the assay described by Gunzler and Flohe46. The assay recording of NADPH loss measures H2O2 reduction by GPx to an alcohol47. To determine the GPx activity within a sample, given that 1 unit = 1 μmole NADPH oxidized min-1 at the specified GSH concentrations or more correctly, μmoles GSH produced min-1; use the following calculations:
GPx activity gels (8% gels) will be green-blue in color with white broad bands where the enzyme is present (approximately 85-90 KDa) (Box 3). Similar to the catalase assay, the in-gel assay determines GPx levels between samples by removal of the reducing agent, peroxide, needed for potassium ferricyanide to ferrocyanide. Removal of peroxide by GPx inhibits the interaction with ferric chloride and thus allows for an achromatic clearing on the gel where GPx is present. With GPx gels, it is critical to run a positive control (such as bovine GPx as demonstrated in Figure 3) as often an upper band will appear corresponding to glutathione S-transferases. Band width will also increase with increasing GPx present in the sample.
In addition to the activity assays and activity gels for SOD, catalase and GPx, the levels of immunoreactive protein for these enzymes can also be determined in cells and tissue by immunohistochemistry (Box 4), immunofluorescence of tissue sections and cultured cells (Box 5 and 6, respectively), and immunogold histochemistry (Supplementary Method).
Wash 107-108, adherent cells three times in 5 ml of phosphate-buffered saline free of Ca2+ and Mg2+ for 30 s (PBS: KCl 2.7 mM, KH2PO4 1.5 mM, NaHPO4 8 mM and NaCl 136.9 mM, pH 7.0). Remove most of the buffer, and then with the aid of a rubber policeman, scrape the cells from the surface of the tissue culture flask. Pellet the cells for 5 min at 200 × g, 4 °C in 1.5 ml microfuge tubes. Remove the supernatant and resuspend the cells in three times the pellet volume in 50 mM phosphate buffer (PB, pH 7.8) and sonicate on ice for a minimum of 1 × 30 sec using a Vibra Cell cup horn sonicator at 40% power. Cell pellets can be stored at -20 °C until assayed. Cell and tissue homogenates can be stored for 6 months at -20 °C. Repeated freeze thaws should be avoided.
Remove animal tissues postmortem. Tissue can be prepared immediately or following storage at -80 °C for up to 1 year. Mince tissue (a minimum of 100 μg) using a razor blade on a glass plate or dish on ice. Place the tissue into a 12 × 75 cm glass tube and add phosphate buffer at a ratio of 1:3. Homogenize the tissue on ice with a motor driven Teflon pestle homogenizer three times for 30 s. Place the sample into a 1.5 ml Eppendorf tube and sonicate for 1 min in 30 sec bursts (with cooling on ice in between) using a Vibra Cell cup horn sonicator at 40% power. All procedures should be carried out at 4 °C. Homogenized tissue can be stored at -20°C for 6 mo.
CAUTION All experiments using animals or human samples should be reviewed and approved by the Institutional animal care and use committee.
Protein concentration for cells and tissue can be estimated by the Bradford method according to the manufacturer’s protocol and standardized using a BSA standard curve (0.25 – 4 μg μl-1). Where indicated, protein concentration is measured by the method of Lowry33. Samples analyzed in 1 ml volumes using water as a control and the absorbance recorded at 595 nm using a spectrophotometer. Cell numbers can also be measured with a hemocytometer or Coulter counter. Enzyme activities can be expressed as normalized per cell, per mg protein or per μg DNA.
Prepare 1 M stock solutions of KH2PO4 and K2HPO4. Dilute 10 ml of 1 M KH2PO4 to 200 ml with ddH2O and 50 ml of K2HPO4 to 1 L with ddH2O. Combine the diluted KH2PO4 and K2HPO4 solutions until the pH reaches 7.8. Store at 4 °C for up to 1 year. During experimentation the PB can be stored at 4 °C or room temperature (25 °C).
Weigh out 0.2635 g of DETAPAC and place into a 500 ml glass bottle, add 500 ml of PB. Prepare fresh every week. Store at 4 °C.
Add 18 mg of xanthine to 100 ml of PB in a 100 ml glass bottle. Loosen the cap, add a small stir bar, and heat to boiling to dissolve. Let the solution cool before use. Prepare fresh every week. Store at 4 °C.
Add 50 ml of PB to a 100 ml glass bottle. Weigh out 0.8087 g of NaCN and add to the glass bottle. Shake to mix. Prepare fresh every 2 d. Store at 4 °C.
CAUTION Sodium cyanide is toxic by inhalation and in contact with the skin. Use in a fume hood and wear gloves
Add 18.3 mg of NBT to 10 ml of PB. Store in an amber glass bottle at 4 °C for up to 1 year.
Add 21.16 mg of BSA to 100 ml of DETAPAC (1.43 mM). Prepare fresh every 5 d. Store at 4 °C.
Add 5.65 mg of BCS to 1 ml of PB in a 1.5 ml microfuge tube and vortex to mix. Prepare fresh every 2 d. Store at 4 °C
Prepare the stock solution by adding ~1 mg of catalase to 1 ml of PB, if the activity for 1 mg equals 20,000 U. Store at −20 °C for up to 1 year.
Dilute the catalase stock solution in PB. Example: Take 200 μl of 20,000 U μl-1 stock and add to 100 ml of PB. Store at −20 °C for 6 months.
Set up two tubes for total SOD and MnSOD assay respectively.
CRITICAL More stock XO may be needed because XO activity will decrease during storage and over the duration of the experiment. Prepare immediately before use and allow a 1 h waiting period for the XO to stabilize its activity. Make fresh daily and store on ice during the experiment.
Add 300 μl of 30% H2O2 (vol/vol, approximately 10 M) to 100 ml of 0.05 M PB in a 100 ml glass bottle. Read the absorbance vs. buffer blank at 240 nm. Adjust the absorbance to between 1.150 and 1.200 by diluting with PB buffer or adding more stock H2O2. Make fresh for each activity assay, the 30 mM H2O2 solution can be at room temperature or 4 °C during experimentation.
CAUTION H2O2 is corrosive and inhalation risk. Protect eyes and skin, (wear goggles, lab coat/gloves, and respirator).
Using PB (50 mM, pH 7.8), prepare a 400 units ml-1 (calculated from the bottle, units mg-1) of bovine liver catalase and store at −20 °C for up to 1 year.
For the monobasic potassium phosphate buffer, weigh out 7.56 g of KH2PO4 and add 0.4136 g of disodium EDTA (Na2EDTA) and 0.0726 g of sodium azide (NaN3), add to 1 L of ddH2O, stir. For the dibasic buffer, weigh out 14.52 g of K2HPO4 add 0.6204 g of Na2EDTA, and 0.1084 g NaN3. Dissolve in 1.5 L of ddH2O. To make the working PB solution, mix by pouring dibasic buffer into the monobasic buffer until the pH reaches pH 7.0 (requires about 1300 ml of dibasic buffer to 1 L of monobasic buffer).
Store at 4 °C for up to 1 year.
! CAUTION Sodium azide is toxic by inhalation and in contact with the skin. Use in a fume hood and wear gloves.
Dissolve 200 units of glutathione peroxidase (GPx) in 10 ml of stock PB (55.6 mM). Aliquot 0.25 ml into 1.5 ml microfuge tubes. Wrap the tubes in parafilm and store at -20 °C for 1 month.
For 50 ml, add 0.0205 g of reduced glutathione (GSH, 1.33 mM) and 0.266 ml of glutathione reductase (GR of 1.33 E.U. ml-1) for 250 U ml-1 add PB to a final volume of 50 ml. [GR calculation: 50 ml × 1.33 E.U ml-1 = 66.5 units / (#units ml-1) = X ml GR to add to buffer]
Prepare fresh on the day of use and store on ice.
Weigh 0.0116 g of NADPH and add to 3 ml of stock phosphate buffer and keep on ice. Make fresh on the day of use.
For determining total GPx activity use cumene hydroperoxide (15 mM) in water, add 116.5 μl to 50 ml of ddH2O or t-butyl hydroperoxide (12 mM) add 83 μl to 50 ml of ddH2O. To determine Se-dependent GPx activity use H2O2 (2.5 mM) as the substrate. H2O2 (2.5 mM) is prepared by dissolving 20 μl of 30% (vol/vol) H2O2 in 50 ml of ddH2O. Read the absorbance of the solution at 240 nm. Dilute with ddH2O until the absorbance is 0.099 [E = 39.4 cm-1 (mol/liter)-1]. Make substrates fresh the day of use and store at room temperature.
Weigh 90.0 g of acrylamide and 2.4 g of bis-acrylamide. Add 280 ml ddH2O to a 600 ml glass beaker, stir with a magnetic stir bar. Adjust the volume to 300 ml with ddH2O. Store at 4 °C for up to 1 year.
! CAUTION Acrylamide and bis-acrylamide are toxic. Protect skin, wear gloves and lab coat, work in a fume hood.
Add 90.85 g of Tris (1.5 M) and 1.49 g disodium EDTA (8.0 mM) to 490 ml of ddH2O. Adjust the pH to 8.8 with concentrated HCl CAUTION toxic by inhalation and in contact with the skin. Add ddH2O to 500 ml. Store at room temperature for up to 1 year
Add 30.95 g of Tris (0.5 M) and 1.49 g disodium EDTA (8.0 mM) to 490 ml ddH2O. Adjust the pH to 6.8 with concentrated HCl. Add ddH2O to 500 ml. Store at room temperature for up to 1 year.
Add 100 mg of APS to 1 ml of ddH2O in a 1.5 ml microfuge tube. Store at 4 °C for 1 month.
Add 20 g of sucrose to a 50 ml conical tube and bring the final volume up to 50 ml with ddH2O. Mix by inversion. Store at 4 °C for approximately 2 months. Visually inspect for gross contamination with microorganisms before use.
Add 2 mg of riboflavin-5’-phosphate to 50 ml of ddH2O in a 50 ml conical tube. Store at 4 °C for up to 1 year.
For 1 L, weigh 22.76 g of Tris and 0.38 g of disodium EDTA. Add to 980 ml of ddH2O, adjust the pH to 8.8 with concentrated HCl, and bring up to 1 L in a graduated cylinder. Make fresh on the day of use and chill to 4 °C.
For 1 L, weigh 6.06 g of Tris, 22.50 g of glycine, and 0.68 g of disodium EDTA. Add to 960 ml of ddH2O, adjust the pH to 8.3 with concentrated HCl, and bring up to 1 L in a graduated cylinder. Make fresh on the day of use and chill to 4 °C.
To prepare 20 ml; add 10 ml of stacking gel buffer (1.5 M, pH 6.8), 10 ml of glycerol, and 200 μl of 5% bromophenol blue solution (wt/vol) in a 50 ml conical tube. Store at 4 °C for up to 1 year.
The recipe below is for two mini-gels that are assembled with 1.5 mm spacers. Commercially available pre-cast Tris-HCl gels can be utilized in place of the recipe below. Prepare and pour immediately before use at room temperature. For SOD gels prepare 12% gels and 8% gels for GPx and catalase protein activity determination.
|Reagent||Volume for an 8% Gel||Volume for a 12% Gel|
|ddH2O||10.95 ml||8.48 ml|
|30% Acyl-Bis (wt/vol)||4.8 ml||7.28 ml|
|Tris separating buffer, pH 8.8||2.25 ml||2.25 ml|
|TEMED||9 μl||9 μl|
|10% APS (wt/vol)||68 μl||68 μl|
This recipe is for a 5% acrylamide stacking gel (wt/vol) and is used with all native gels. The riboflavin is utilized as a source of free radicals, in the presence of light, to catalyze the polymerization of the gel. This is used in replace of APS due to the potential enzyme inactivation that APS could cause. This also has the added advantage that the acrylamide does not polymerize until activated by light exposure. While the riboflavin/sucrose/acrylamide stacking gel is optimal, a conventional stacking gel containing ammonium persulfate can be utilized because the entire gel, including the stacking gel, is pre-run in buffer removing free persulfate ions from the gel that can potentially inactivate the antioxidant enzymes to be measured. Prepare and pour immediately before use at room temperature.
|30% Acyl-Bis (wt/vol)||1.0 ml|
|Tris Stacking Buffer, pH 6.8||1.6 ml|
|40% Sucrose (wt/vol)||3.2 ml|
|0.004% Riboflavin-5’-phosphate (wt/vol)||800 μl|
Add 40 ml of ddH2O or phosphate buffer into a 50 ml conical tube, add 80 mg of NBT (2.43 mM), 170 μl of TEMED (28 mM) and 8 μl of stock riboflavin-5’-phosphate (0.14 M [53 mg ml-1] in 50 mM phosphate buffer, pH 7.8). Make fresh daily and use at room temperature.
Add 40 ml of ddH2O or phosphate buffer into a 50 ml conical tube, add 80 mg of NBT (2.43 mM), 170 μl of TEMED (28 mM) and 8 μl of stock riboflavin-5’-phosphate (0.14 M [53 mg ml-1] in 50 mM phosphate buffer, pH 7.8) and 1.44 mg of NaCN. Make fresh daily and use at room temperature.
Weigh out 2 g PFA in a fume hood, set aside. Combine 5 ml PBS (10X) and 25 ml water in a 250 ml beaker. Heat the solution for 30 sec in microwave oven. Add the PFA and mix (using a stir bar). Add 1 ml NaOH (5N) dropwise with a 5 ¾” glass Pasteur pipette and 2 ml bulb and mix until dissolved. pH to 7 with HCl. Bring the total volume to 50 ml. Cool on ice or 4°C. Store for 1 week at 4°C in the glass beaker covered with Parafilm or 100 ml glass bottle. Make fresh weekly.
! CAUTION PFA is toxic by iinhalation. Use in a fume hood and wear gloves.
Add 3 ml of 30% (wt/vol) H2O2 to 45 ml of ddH2O in a glass beaker containing a stir bar and mix. Make fresh each time of use and store at room temperature.
Add 2 drops each of A and B to 5 ml of PBS. Make 30 min prior to use. Keep at room temperature, make fresh daily.
Add 1 drop of chromogen to 1 ml of chromogen diluent. Keep at room temperature, make fresh daily.
Add 10 ml of Harris Hematoxylin using a pipetter and a disposable 10 ml plastic pipette into a glass beaker containing 90 ddH2O. Gently mix using the pipetter. Keep at room temperature for up to 1 year. Filter before use if not prepared fresh.
Add 1 ml of concentrated ammonium hydroxide to 100 ml of ddH2O to prepare a 1% (vol/vol) ammonium hydroxide water solution. Keep at room temperature, make fresh daily.
Mix together 200 μl of donkey serum (2%, vol/vol), 0.1 mg of BSA (1%, wt/vol), 10 μl of Triton X (1%, vol/vol), 5 μl of Tween-20 (0.05%, vol/vol), and 10 ml of PBS (0.01 M). Store at 4°C, make fresh weekly.
Add 100 ml formaldehyde (4%, wt/vol), 18.6 g monobasic sodium phosphate, 4.2 g sodium hydroxide into a 1500 ml beaker containing 970 ml ddH2O and a stir bar. pH to 7.2-7.4 and bring volume to 1000 ml with ddH2O. Store at 4°C for 1 month.
CAUTION Formaldehyde is toxic. Protect skin, wear gloves and a lab coat.
Prepare the following two solutions: Add 27.6 g sodium phosphate, monobasic to 1000 ml ddH2O in a 1500 ml beaker. Add 28.4 g dibasic sodium phosphate into a 1500 ml beaker containing 1000 ml ddH2O and stir. Keep each solution at room temperature for up to 1 year. Mix the two solutions in the following ratios: 19 ml of solution monobasic buffer and 81.0 ml of dibasic buffer with 100 ml of ddH2O. This gives a final volume of 200 ml of a 0.1 M solution.
Add 6.1 g of Tris (0.05 M) and 9 g of NaCl (0.9%, wt/vol) into a 1500 ml glass beaker containing 980 ml ddH2O and stir. pH to 7.6 and bring the final volume to 1000 ml with ddH2O. Store each solution at room temperature for up to 1 year.
Add 4 g of BSA (4%, wt/vol) and 500 μl Tween 20 (0.5%, vol/vol) into 100 ml of Tris-buffered saline (0.05 M Tris with 0.9% NaCl (wt/vol), pH 7.6) and mix in a 250 ml glass beaker containing a stir bar. Make fresh daily and use at room temperature.
Dilute the blocking buffer 10 fold. Make fresh daily and use at room temperature.
|Solutions||Final Concentrations||Volume required for SOD System|
|PB with DETAPAC and BSA||0.05 M PB, 1 mM DETAPAC, 0.13 mg BSA||12.9 ml|
|Catalase 40 U ml-1||1 U||0.5 ml|
|Xanthine 1.18 mM||100 μM||1.7 ml|
|NBT 2.24||56 μM||0.5 ml|
|PB||0.05 M||0.3 ml|
|BCS 10 mM||50 μM||0.1 ml|
|Total volume||16.0 ml|
|Solutions||Final Concentrations||Volume required for MnSOD System|
|PB with DETAPAC and BSA||0.05 M PB, 1 mM DETAPAC, 0.13 mg BSA||12.9 ml|
|Catalase 40 U/ml||1 U||0.5 ml|
|Xanthine 1.18 mM||100 μM||1.7 ml|
|NBT 2.24||56 μM||0.5 ml|
|NaCN 0.33 mM||5 mM||0.3 ml|
|BCS 10 mM||50 μM||0.1 ml|
|Total volume||16.0 ml|
|Dilution||Volume of SOD||Volume of PB|
|Dilution #1 (1:1000) (Final concentration 2-10 ng/tube).||1 μl||999 μl|
|Dilution #2 (1:100) (Final concentration 25-50 ng/tube)||3 μl||297 μl|
|Dilution #3 (1:10) (Final concentration 500 ng/tube).||5 μl||45 μ|
|→||absorbance at 60 s|
|→||total mls in reaction; both blank and sample|
|→||total mls of sample; both blank and sample (example if 20 μl of sample was added before splitting the sample in half, denominator = 0.02 ml)|
|→||divide by the protein concentration of the original sample|
|Standard Concentration (U ml-1)||1 U ml-1 GPx (μl)||Stock PB (μl)|
|Phosphate buffer (PB)||50 mM|
In most cell lines, total SOD activity can vary from 20 units mg-1 protein to 90 units mg-1 protein. The total SOD activity consists of 10 to 50 units mg-1 protein of MnSOD and the remaining SOD activity would be attributed to CuZnSOD15. Catalase activity in the same samples can vary from 5 to 30 mk mg-1 protein and GPx runs from 14- 30 units mg-1 protein. However, with gene transfection of the various antioxidant proteins with either stable transfections or infection with adenoviral vectors containing the cDNA for the various antioxidant proteins, activity can increase up to 30-fold compared to baseline values16,17. With activity gels (Fig. 2 and and3),3), specific protein activity cannot be determined, however native gel densitometry is often used to determine a relative activity content which also correlates well with immunoreactive protein and subsequent activity assays15,16,17. Antioxidant immunofluorescent staining of cultured cells (Fig. 4) and immunogold techniques (Fig. 6) are also beneficial in not only determining increases in the antioxidant enzyme that is overexpressed, it also gives potentially valuable information regarding cellular localization. Immunohistochemistry for antioxidant proteins can demonstrate changes in many disease states28. For example in Figure 5, pancreatic cancer specimens demonstrate significant fibrosis and an increased inflammatory cell component to some of the histological sections. Fibrotic areas and inflammatory cells should be excluded in any region of interest because their dark staining falsely increased the measured staining intensity. As seen in Figure 5, strong staining is seen in the cytoplasm in cells from normal pancreas, while staining is nearly undetectable in cells from pancreatic cancer resections with a marked decrease in the mean gray level value when compared to normal pancreas.
This work was supported by NIH grant CA137230 and a VA Merit Review grant.
Competing Financial Interests The authors declare that they have no competing financial interests.