In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.
In this video, we have described the extraction of mitochondrial protein complexes from human embryonic stem cells, their separation by high resolution clear native gel electrophoresis (hrCNE), and the subsequent analysis by in-gel catalytic activity assays. hrCNE resolves mitochondrial membrane protein complexes at a resolution comparable to that of blue native gel electrophoresis and is superior for in-gel activity assays. This technique can be employed not only to quantify mitochondrial respiratory complexes I-V but a…
Human embryonic stem cell studies in the Teitell lab are currently supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank Dr. Carla Koehler (UCLA) and members of the Koehler laboratory for providing advice and insights in these and additional studies.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
0.5M EDTA | Fisher Scientific | BP120-1 | adjust pH to 8.0 with NaOH | |
1M NaCl | Sigma | S3014-500MG | ||
1M Sodium succinate | Fisher Scientific | S413-500 | ||
0.1M Sodium phosphate buffer pH 7.2 | Fisher Scientific | BP332-1 and BP329-1 | Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C | |
1M Tricine, pH 7.0 (adjusted with NaOH) | EMD | 9010 | store at 4 C | |
1M imidazole/HCl, pH 7.0 | Sigma | I-2399 | store at 4 C | |
1M Tris-HCl, pH 7.4 | Fisher Scientific | BP153-1 | ||
20% (wt/vol) dodecyl-b-D-maltoside (DDM) | Fluka | 44205-500MG | Dissolved in water. Store 1 ml aliquots at –20 C. | |
10% (wt/vol) digitonin (>50% purity, used without recrystallization) | Fluka | 37008-1G | Dissolved in water. Store 0.1–1 ml aliquots at –20 C. Should be warmed up to 95 C before use. | |
2M 6-aminohexanoic acid | Fluka | 07260-100G | store at 4 C | |
10x Ponceau S/glycerol stock solution | Sigma | P-3504 and G6279-1L | 0.1% Ponceau S, 50% glycerol, (wt/vol) in water. | |
10% w/v sodium deoxycholate | Fluka | 30970-25G | Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature. | |
250 mM phenazine methasulfate (PMS) | TCI America | P0083 | Dissolved in DMSO and aliquoted. Store at –20 C. | |
Nitro-blue tetrazolium (NBT) | Amresco | 0329-1G | ||
3,3’-diaminobenzidine tetrahydrochloride (DAB) | TCI America | D0078 | ||
Pb(NO3)2 | Fisher Scientific | L62-100 | ||
MgSO4 | Fisher Scientific | BP213-1 | ||
Tris base | Fisher Scientific | BP152-10 | ||
Glycine | Fisher Scientific | G45-212 | ||
Adenosine 5’-triphosphate disodium salt (ATP) | Sigma | A2383-5G | Grade I, minimum 99% | |
beta-nicotineamide adenine dinucleotide (NADH) reduced form | Sigma | N-8129 | ||
Cytochrome c From horse heart | Sigma | C2506-250MG | ||
Sucrose | Fisher Scientific | BP220-212 | ||
100x protease inhibitor cocktail | Sigma | P8340 | Store at – 20 C. | |
Dimethyl sulfoxide (DMSO) | Fisher Scientific | D128-500 | ||
Trypan blue Stain 0.4% | Gibco | 15250-061 | ||
10x Trypsin (0.5%) | Gibco | 15400-054 | ||
Trypsin Inhibitor | Gibco | 17075-029 |