Lab Protocols

Procedure: Utilize the following mix components for all experiments. This protocol has been optimized for transfection of neonatal rat cardiac myocytes. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Add 40 μl per well immediately after plating (20 μl each of the luciferase plasmid with 20 μl of the beta gal mix). All mixing (except that which requires vortexing) should be done in the sterile cell culture hood. Total volume 400 μl 600 μl 800 μl Sterile 2.5 M CaCl 2 25 μl 37.5 μl 50 μl Plasmid 10 μg 15 μg 20 μl Sterile H 2 0 Total of 200 μl when added to above. Bring to total of 300 μl with sterile H 2 0 Bring total to 400 μl with H 2 0  Combine the above three reagents in a hood using a sterile 1.5 ml Eppendorf tube. Mix gently. Do not vortex. Sterile 2X HBS (hepes buffered sa...

Considerations for use The principle of the biuret assay is similar to that of the Lowry, however it involves a single incubation of 20 min. There are very few interfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much more material. The biuret is a good general protein assay for batches of material for which yield is not a problem. The Bradford assay is faster and more sensitive. Principle Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. Equipment In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximu...

Considerations for use The bicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace the Lowry because it requires a single step, and the color reagent is stable under alkaline conditions. Both a standard assay for concentrated proteins and a micro assay for dilute protein solutions are described below. Principle BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under ...

Considerations for use See considerations listed under the absorbance assay at 280 nm. This method is just as convenient as for absorbance at 280 nm. It may be preferred if there is excessive contamination by nucleic acids, since nucleic acids absorb very little radiation at 205 nm. Setting the wavelength is a bit tricky since 205 nm is right on the shoulder of the protein peak. Principle See the discussion for the 280 nm absorbance assay. Equipment In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required. Procedure Include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces. This is necessary for the 205 nm assay because losses are proportionately higher ...