I wanted 5uG in each lane.
For Lane 2 I had a concentration of 1.08mg/mL = 1.08ug/uL. 5ug x 1uL/1.08ug = 4.63uL. I mixed (4.63uL x 3) with the same volume of laemlli sample buffer to get a total of 27.78uL which contains 15ug of the protein. Then I loaded 1/3 of the total sample (27.78uL / 3 = 9.26uL).
What can help me to get sharper bands as well as remove the smearing?
In addition to Monika and Tamás comments, you can use SDS Gradient PAGE gel. Bands in SDS gradient gel are usually sharper than in homogeneous SDS PAGE gel.
Andy, sharpness of band depends on many things in addition to the the conc. of loading sample. e.g glycoproteins generally gives the bloch instead of sharp band.
On looking your SDS PAGE figure i think you have over loaded, because of which it is forming a smear.
If you want to load 5ug in each lane you can do following calculation (sorry i couldt understand your calculation):
As your protein load is 1.08mg/mL
so you should mix 10uL of this sample in 30uL of laemlli sample buffer and load about 19uL of mixture in well, you will be loading 5.13uG of your protein in this case.
If I were you I d use additional SDS in the Sample Buffer and/or in the PAGel. I worked for me. I always make gels with a higher cc. of SDS in the stacking if there are smears. On the other hand always use proteinase inhibitors for unwanted proteinases. Good luck!
In addition to Monika and Tamás comments, you can use SDS Gradient PAGE gel. Bands in SDS gradient gel are usually sharper than in homogeneous SDS PAGE gel.
Did you boil your samples? over boiling could cause smear bands because of complete denaturation of proteins. use more samples + more sample buffer in max 5 min in 95 C. if the problem remained, there is a problem prior to these steps with your protein samples integrity, like wrong protein extraction or protease contamination.
Please tell us about your experience.
I agree with Monica comment. I guess u have overloaded the protein. Quantitate ur sample without any errors. Load the concentrated protein samples mixed with 5 volume of lamelli buffer(for example 1 microliter of protein with 15 microlitre of lamelli buffer)
Simply include 2mM iodoacetamide from Biorad or Sigma in your sample buffer, use sigma or SRL or invitrogen or BioRad acrylamide, run at low voltage with freshly prepared 1X running buffer and good quality resolution is assured.
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