Problem with SDS-PAGE - (May/04/2006 )
i am trying to expressa mutant protein of 15 Kd in pichia and i collect the samples during 4 day growth and analysing them on SDS-PAGE( using 18% biorad gels n gel code blue stain). i prepare the SDS samples by taking 20 microliters of lysate, 20 mcl of 62.5mM tris-hcl pH 6.8, 10 mcl 05 10mM SDS,4-6 mcl of BME and boil for 4 min, cool and 54-56 mcl of 20 % glycerol, 10 mcl of bromophenol blue.and i load 20 mcl of this sample in th wells. and run the gels at 150 V untill the dye front reaches the bottom of gel. the problem is some times gels have faint bands corresponding to the protein of interest and some gels doesnot show any band. i am trying get atleast one good gel so that we can send it to Mass spec.
can anyone suggest what i should be doing.
thanks in advance.
Could this be a concentration issue? Try concentrating your protein.
We used a similar method to yours but had an affinity chromatography stage and a centrifugal concentrating stage. Discuss with the mass spec lab.