In my work, I extracted proteome from breast cancer cell line by lysis buffer and precipitated it with acetomethanol. I use 180 µg of proteins and I have separated it on a 12% sds-page. The running buffer that I used was consist of 25mM Tris, 192mM glycine, 0.1% SDS and my apparatus is from Bio-rad company. What do you think about my gel? How can I improve horizontal streaking?
This can be said a nice 2D gel if you are a beginner. The horizontal striking arises due to improper IEF, impurities in sample or protein degradation. Your gel suggests two major problems.
The first issue is with sample preparation, as you used methanol, which resulted in nucleic acid contamination in your protein sample due to which most of your spots get covered by black patched. I think you can get rid of this problem by using extracting your protein in tris-saturated phenol and precipitation it with chilled methanol (with 0.1 M ammonium acetate). The second issue is with IEF step. Most of the spot in your gel are comet-shaped, suggesting incomplete focusing. You need to increase the KVH of IEF step. Better If you mention the length of your IPG strip, KVH of IEF and the running condition of second dimension.
thanks indeed Mr. Ambashtha for your consideration
my IPG strip length is 17cm.
IEF condition:
Rehydration: 50v-18hr
S1: 200v-1hr-slow-desalting
S2: 4000v-2hr-linear- focusing
S3: 8000v-4hr-linear- focusing
S4: 10000v-1hr-linear- focusing
S5: 10000v-80Kvh-rapid
Second dimension condition: 16 mA/gel, 30min-24mA/gel, 6hr
could you explain more about trees-saturated phenol method?
Rehydration is little long , you can do passive rehydration 0/N at 200C by covering with mineral oil, or 50v - 2hr will be sufficient. your focusing condition is adequate . I get better result using fixed voltage in 2nd dimension. I used 100v for 10- 14 hours and used 2X buffer in upper tank. Sending the link of phenol extraction protocol.
https://www.dropbox.com/s/vgx6gvar1dpdmnt/Total%20proteins%20extraction%20buffer%20for%202-D%20RG.pdf?dl=0
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