I tried protein precipitation with TCA (250ul in 900 ul of sample volume) and washed with Acetone 4 times..the pellet is not completely dissolved in the sample buffer.
1. I put it on the hot plate at 95C for 5mins, the pellet does not dissolve with sample buffer even if I vortex.
2. My target protein in that sample is of 21kDa..Can we use sonication and collect the supernatant for loading in SDS gel?
I would suggest you to try to vortex your pellet with sample buffer first before putting it onto the hot plate and add more sample buffer to dissolve it. Normally when I encounter TCA precipitation, I will load 60uL of 1X sample buffer to the pellet in a eppendorf and have no trouble dissolving with various recombinant protein(whether soluble or inclusion body)
As to your second point of view, I think its fine but you need to make sure the protein as a high enough concentration in the SDS-PAGE for western or do a TCA precipitation after sonication. As for me, my protein's signal is way to high on Western after sonication then TCA precipitation.
I hope this helps. It's my first time answering question. So if anything is unclear, please do let me know. Thanks.
The capacity of the sample buffer to dissolve the protein pellet is not unlimited. If there is too much protein in the pellet, a larger volume of sample buffer will be needed to dissolve it.
We encounter this problem often after TCA. A way that works well for us is bath sonication in sample buffer for 2 hours. This avoids diluting your protein too much. If you do this on a regular basis, you may consider sample buffer containing Urea/Thiourea, more efficient for pellets than SDS only. I hope this helps
We use sample buffer with 4M urea and this helps a bit to solubilize protein pellet after TCA precipitation and Acetone wash.
I encountered the same issue when I precipitate total protein from fungus using 20% TCA. Even I heated protein at 100 degrees for 10 mins with intermittent tapping/vortexing. I did not face the same issue when I precipitate protein with acetone! adding more sample buffer to it would dilute total protein that is another concern. Maybe you can try acetone or methanol precipitation..
Yes, this type of problem may occur when you precipitate protein via methanol (I have faced this issue while I was precipitating protein after Click IT chemistry). The best solution I came up with is the dissolving protein palette in a high volume of sample buffer or RIPA whatever you use. Vortex it for 5 min. Give 2-3 cycles of 15 min in a bath sonicator. This will definitely increase the yield.
Suggest some buffer in which protein ppt of human serum should be dissolved or stored
Suggest some buffer in which protein ppt of human serum should be dissolved or stored
You can store it as a solid, with no buffer. What you use to dissolve it will depend on how it was precipitated and what you want to do with it afterwards.
Thank you so much for response.
I ppt it by TCA acetone method, for sds i dissolved in loading buffer. For long term storage in which buffer it should be dissolved.
Thank you so much for response.
I ppt it by TCA acetone method, for sds i dissolved in loading buffer. For long term storage in which buffer it should be dissolved.
It should be OK to store the pellets or the samples in loading buffer in the freezer for a long time.
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