Sample buffer

Go to the profile of Chinmaya Sadangi
Feb 06, 2018
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5

Hi,

I have 800 µl of sample buffer stocks (without DTT) stored at -20°C. Before using I add 2.5 M DTT and use it.

Sometimes, I add the sample buffer to my samples and store at -20°C. However, the next day after heating the samples, the color of the buffer changes from blue to dark green. But, when I load it to the gels, they appear blue. Why does the color change after heating.

After adding the buffer to the sample, should I store the buffer at -20°C for next day use or should I store at RT. How long can the buffer be stored (days, weeks?) at RT or -20°C?

Thanks.

5 Comments

Go to the profile of relaxin
relaxin 5 months ago

I do not recall any change of color of samples after heating. It could be due to change in pH. I guess it will not affect the sample.

When you add sample buffer to your samples, you need to boil them immediately. Some proteases are active in the presence of SDS. After boiling, the samples should be safe for storage at 4 C overnight. For longer term storage, they should be stored at -20 C. You need to reheat them again prior to loading on gel. This is to make sure everything is re-dissolved.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

So, you suggest heating the sample twice.
If stored at 4°C overnight, do I need to re-heat it?

Go to the profile of relaxin
relaxin 5 months ago

The second heating can be done at 70 C for 2 min. For samples stored overnight at 4 C, SDS may form precipitate, heat at 70 C will re-dissolve it.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

I am using KCC2 antibody and it has been advised not to heat more than 37 degrees. So do you suggest heatijg it again at 37 degrees for 30 min after incubation at 4 degrees?

Go to the profile of relaxin
relaxin 5 months ago

I think the advice of not to heat the antibody over 37 C is to preserve its function. When you analyze your IP on SDS gel, boiling the sample is to destroy the antibody and release the antigen. I do not think you need to use low temperature.

Of course, the second heating can be done at 37 C, since this is to re-dissolve SDS.