<10kD protein in Western - (Jul/08/2005 )

Hi I need to probe a very small protein (<10kDa) by western. I used millipore Immobilon pSQ and transfered 1hr (semi-dry), it didn't work. Unfortunately I don't have control so that I actually don't know if it is the problem of the membrane, antibody or amount of the protein. Do anyone have experience with such a small protein and have some tips to share with? Thanks!

---

Hi. I blotted 7kDa protein on Serva NC45 nitrocellulose membrane (pore size 0.45 micrometers) (semi-dry transfer (MeOH, glycine + SDS in buffer) for 1 h, approx. 35 mA for normal size gel (8×5 cm)). It worked very well.

---

Hi mpblue

Thanks for the reply. What blocking solution (milk or BSA) do you use? Does you wash buffer contain tween20?

---

Sorry for late reply. For handling the membrane after transfer I use PBS+0.05% Tween-20 (in all steps). For blocking I add non (or low) fat milk (5%).

I prepare the transfer buffer as follows: 2.93 g Tris, 5.81 g glycine, 3.75 ml 10% SDS --> add water to 800 ml --> dissolve --> add 200 ml of methanol.

I hope this works for you.

---