Labeling Buffer

Solution I 625 III of 2M Tris HCl (pH 8) 25 pi 5 M MgCl2 5 pi each of 100 mM (pH 7) dATP, dTTP, dGTP (Pharmacia or Promega) 18pi of 2-P-mercaptoethanol, 350 pi dH20. Solution II 2M HEPES titrated to pH 6.6 with NaOH, filter sterilized, and stored at 4°C. 

Combine solutions I, II, and III in the ratio 2 5 3 and freeze in aliquots of 50 pi. The resultant solution (designated labeling buffer) can be freeze thawed at least two or three times and is stable at —20°C for at least 1 year. 

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To each ml of glycoprotein solution, add 30 pi of neuraminidase (1 unit/ml as supplied by Behringwerke AF), then 30 pi of galactose oxidase (previously dissolved at 100 units/ml in the labeling buffer of step 1), and finally 100 pi of the biocytin hydrazide solution. 

Labeling buffer PBS supplemented with 0.5% bovine serum albumin and 2 mM ethylene diamine tetraacetic acid. 

Wash cells twice with labeling buffer by centrifugating at 300g and proceed to determine cell concentration using a particle counter. Also, perform a viability test using a Trypan blue exclusion method. Keep cells in cold storage (4°C) until used. 

Assemble the membrane holders with the polycarbonate membrane previously moistened with labeling buffer, attach syringes, and mount in a syringe pump. Set flow rate to a maximum of 1 mL/min (see Note 9). 

Resuspend the cell pellet in labeling buffer (about 5 mL final total volume) and proceed to count recovered cells in triplicate (see Note 7). An automated particle counter is highly recommended because it is possible to set size gates for cancer cells, erythrocytes, and leukocytes. 

The use of trypsin is common to detach cells from adherent cultures however, in this case it is not recommended. MCF-7 cells treated with trypsin tend to form clumps, which are difficult to breakup. The use of Accutase yields a mostly single-cell suspension that will not form clumps even after it has been washed with labeling buffer. Total cell count and viability assays will also be easier for the researcher to carry out. 

To avoid contamination by DNases, it is advisable to sterilize the solutions used in the TUNEL/ISEL procedure, particularly the labeling buffer, and to use sterile pipet tips and microcentrifuge tubes. 

Terminal deoxynucleotidyl transferase (Gibco BRL, Grand Island, NY). Labeling buffer (5X 500 mM potassium cacodylate, pH 7.2, 1 mM DTT). Store at -20°C. 

Standard 40 pL reaction mix 1 pL scissile strand oligonucleotide (approximately 10 picomoles) with 20 pL purified distilled water, 8 pL 5X labeling buffer, 10 pL [a32P]-cordycepin and 1 pL terminal deoxynucleotidyl transferase (TdT) (15 units). 

Pre-labeling buffer It is the wash buffer without imidazole in it. 

Native Extract from HEK 293 cells (Ab Array Extraction/Labeling Buffer)... 

Fig. 1. Western analyses of extraction efficiency of the antibody microarray extraction/labeling buffer. Equal amounts of sample extracted by the extraction/labeling buffer and by sodium dodecyl sulfate (SDS)-boiling method (1% SDS in TST buffer for 5 min at 100°C) were separated by using SDS electrophoresis in twelve lanes (total of 20 pg per lane) each and each lane for both extracts was probed after a transfer of the proteins to a Polyvinylidene Difluoride (PVDF) membrane with a cell compartment-specific marker antibody. Lanes are 1. MW Standards, 2. Empty, 3. Vinculin— Cytoskeleton, 4. Empty, 5. ERK 1—Cytosol, 6. BIP/GRP78— Endoplasmic reticulum,...

While holding the pestle over the mortar, rinse the pestle with 1-2 ml of extraction/labeling buffer. 

Dilute each sample to 1.1 mg protein/ml by adding the appropriate volume of extiaction/labeling buffer. The final volume must be >1 ml (see Note 4). 

Dissolve the Cy3 dye in 110 pi of extiaction/labeling buffer by adding the buffer directly to the tube in which the dye is supplied. Note Each tube of dye contains a quantity of dye that will label approximately 1 mg of total protein. 

Weigh the tissue sample in the mortar (pre-weigh the mortar to determine the exact amount of the tissue sample) before adding the buffer Extraction/labeling buffer is added at volume/weight ratio of 20 pl/mg of tissue. 

Ab Microarray Buffer Kit (Cat. No. 631792, Clontech, Mountain View, CA, USA) containing, Incubation Tray, Extraction/Labeling Buffer, Stock Incubation Buffer, Background Reducer, and Wash Buffers A-C. 

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