ABE Workshop 2006. The SDS-PAGE. Dongping Lu. Western blotting. Trouble shooting. 1. We should have a parameter: Total protein /plant tissue 2. Use higher concentration of BSA Standard, like BSA 10mg/ml. Absorption value. BSA. Today’s work. Run the SDS-PAGE gel:
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1. We should have a parameter: Total protein /plant tissue
2. Use higher concentration of BSA Standard, like BSA 10mg/ml
group 1 & 4: wt and pdi2 mutant
group 2 & 3: wt and gfp-2sc
1. SDS disrupts some of the noncovalent interactions that stabilize protein quaternary and tertiary structures, facilitates denaturation.
2. SDS also has a negative electrical charge and binds to proteins in a constant mass ratio of 1.4 : 1, so that the total amount of detergent bound is directly proportional to the molecular weight of the protein.
3. The ‘coating’ of negatively charged SDS overwhelms the inherent charges of protein molecules and gives them a uniform charge to mass ratio.
4. This allows proteins to be separated on the basis of their relative sizes,SDS
lower concentration of acrylamide (larger pore size),
different ionic content