Western Blotting help - (Aug/18/2008 )

Hi I have several questions as I am new to Western blotting.

I am trying to detect a 35kDa protein in gram negative bacteria E. coli and Legionella. My primary antibodies were made in rabbits. I use PVDF membrane.... and yes I know about pre-soaking it in 100% methanol, as I already made that mistake to not do it.

1) Should bacterial pellets be boiled at 100 degrees Celcius for 10 minutes in Laemmli buffer + 540mg/ml DTT (1:10 in Laemmli)?

2) Can I use fetal calf serum (5%) in PBS with 0.3% tween-20 for blocking?

3) To purify my primary antibody, I want to use a Protein A column. Are they suppose to be really slow? How can I speed this darn thing up?
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Thanks!

Sounds like you'll be having lots of fun with this.....

1) Generally use 1x Laemmeli sample buffer, not 1:10 dilution. Also you will need to do a protein estimate, so that you won't overload your gel. For mini-gel I would load maximum 20 ug per lane.

2) No, use 5% non-fat milk powder (you can buy from supermarket).

3) How much serum did you load? Dilute your serum (1 ml) in PBS (9 ml). It's better to affinity purify against your antigen, so you get your specific antibody, not all the IgG (as from Protein A) column.

Good luck.