Hello, I am currently confused with a few following steps in BSA denaturation inhibition assay.
1. Some protocols stated that the buffer was added along with BSA and tested solution before incubation of 37°C. Whereas, the others added the buffer after cooling off the mixture. Which is the correct way?
2. After incubating at 37°C in 20 minutes, should I take the mixture out of the water bath and wait for it to heat up to 72°C or keep the mixture inside the water bath until 72°C? I only have 1 water bath
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1. Some protocols stated that the buffer was added along with BSA and tested solution before incubation of 37°C. Whereas, the others added the buffer after cooling off the mixture. Which is the correct way?
You need to add the buffer along with BSA and the test solution and then incubate at 37 degree C for 20 mins.
2. After incubating at 37°C in 20 minutes, should I take the mixture out of the water bath and wait for it to heat up to 72°C or keep the mixture inside the water bath until 72°C? I only have 1 water bath.
You cannot keep the mixture out waiting for the water bath to reach 72°C or keep the mixture inside the water bath until 72°C because it may take around 20-30 mins for the water bath to reach 72 degree C. Since you do not have another water bath, there are two ways which you could follow:
a. If you are friendly with the neighboring laboratory, you could always request them for the water bath to be set at 72 degree C.
b. Another way would be to use the incubator (if you do have one) set at 37 degree C. Place the glass container (beaker) containing water at 37 degree C in the incubator. Use this container to immerse the tubes. Check the temperature of the water in the container manually with the thermometer. At the same time set the water bath at 72 degree C.
After cooling for about 20 mins you may measure the turbidity of the solutions at 660nm using the spectrophotometer.
Best.
According to my literature survery and experiment, make BSA in buffer. And make up the volume of sample with buffer.
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