Some lanes see bands,other no, strange! - western blotting questions (Sep/27/2005 )

sometimes, in my western blotting result(in film), some lanes show interesting band(usually in middle lanes of PVDF, other lanes show no bands(on the two rim of lanes). But it should all have bands, even one time , I load the same sample with same level, but one proximal to the rim lane show no band, the other show strong signal band.
Anyone have ever happened ?
Anybody sees it, could you give your explanations ?
Thanks anyone

---

Do you have a MW marker in the outer lane(s) and is transfer of it occurring?

How are you doing your transfer/blocking/primary Ab/secondary Ab steps and washings? Are you doing any of this in tubes?

---

Yes, I run the prestained MW marker in the outer lane, but it stay on the PVDF.

I do transfer/blocking/primary Ab/secondary Ab steps in the hybridization bag, wash it in small tanks. Does it change for better in tubes?

Thanks
waiting for your details

---

I don't think that your waashing steps are the problem but rather the transfert of your proteins, do you use semi dry transfer ? I would not judge too much a transfert via the MW marker specially if you use a prestained

Pesji

---

I think it might be the way you are packing your transfer sandwich. Make sure the sandwhich is tight. You have to put an equal pressure on the membrane and make sure that all the parts od the membrane is in contact with the gel. Try and use 2 pieces of filter paper or an extra sponge.
Also make sure that the sandwhich is always wet with transfer buffer.
good luck
p..

---

I am sure the transfer process is no problem, because the same PVDF after exposure to x-film is dyed with all bands .
does anyone think that the primary or second antibody is not evenly mixed and combines to PVDF?
Thanks

---

Incubate your primay and secondary antibody in more TBS buffer so that the buffer cover the whole membrane.
Make sure your container that is use for incubating your membrane is flat. If not, the antibody may not be able to attach to bands on the edge of the membrane.
Put the container with the membrane on a rocker or shaker.

---

Thanks
I incubate the membrane and Ab in hydridization bag not container or tank.
somebody tell me that the 50ml centrifuge tube is better to use to incubate the membrane and antibody mix than the hydridization bag. is there anyone in favor of it?

---

Thanks
I incubate the membrane and Ab in hydridization bag not container or tank.
somebody tell me that the 50ml centrifuge tube is better to use to incubate the membrane and antibody mix than the hydridization bag. is there anyone in favor of it?
[/quote]

Well this trick is mainly used to limit the amount of antibody to be used, the good thing is that the solution gets more uniformely onto the membrane during the rolling. But that will not change your strange bands problem I'm afraid !

pesji

---

I use the tube method and it works OK most of the time.

I get missing lanes, or half lanes at the far end of my membrane, but never at the marker end.

I'll aslo somtimes get a lovely result for the first four samples, with beautiful strong bands and lovely beta actin, then a horrible patche spotty, weak and feeble for my second four samples.

---