western blot - (Mar/06/2001 )

I'm currently perfoming western blots on a rather large protein (approx. 400 kD).My problem is two-fold: First, I've read that in blotting larger proteins, the methanol content should decrease. I've also read that the time of electroblotting should alsoincrease. For a protein this size, what should time and methanol content parametersbe (a good starting point)? Second, I use HRP conjugated secondary Abs in whichI use ECL as a detection reagent for chemiluminescence. My blots have highbackgrounds with spots on them. My troubleshooting efforts include the following:increased wash cycles (4xs @ 15 min each wash w/20 ml washing buffer each wash),playing around with Ab conc (secondary=1:1000, 1:1500; 1:3000, all producingsimilar results), new PVDF membrane, remaking all reagents and checking pH,thoroughly cleaning glass plates before I pour and run the SDS-PAGE. I also blockin TTBS/fat-free milk (5%) overnight at 4 degrees C. Any suggestions of what else can be done outside of these that I just mentioned? Any suggestionsare appreciated.Thanks,Tracey

I'm not sure but we use our secondary antibody at 1:10,000 dilution. Using a higher amount may explain the higher background you observe.

I hope this helps. Good luck.

Have you tried an acetone precipitation? It works on plant extracts. Or a high-saline buffer system? Adding PMSF to protein extracts sometimes improves the blots. During an over-night incubation with milk-powder 0.02% Sodium azide could be added.

The methanol is only useful for binding to nitrocellulose membranes. If you use PVDF membranes (which bind proteins muchstronger anyway) you can leave out all the methanol (so use zero % ). This will increase the transfer of large proteins, since themethanol tends to shrink the gell and its mesh/pores thereby decreasing especially the transfer of larger proteins.I would recommend using 1 % normal serum from the same species as your conjugate and 0,1 % tween in all your westernblot solutions. Also block with 1% of this serum and 0,1 % tween Overnight at 4 degrees.Normal animal sera are ten-times cheaper at Gibco compared to Dako. 100 ml only costs about 17 dollars.I'm not a Gibco Life Techn. representative.

Happy blotting !Jeffrey.

i am grad student and i want to to do RT-PCR from rat heart tissue. can anyone recommend a kit/protocol/website i can glean some infothanks