Gap43 protein western Blot - (Feb/16/2008 )

Hello!
I'm new in this forum...
I've working with neuroblastoma cell lines and i'm doing a western blot with proteic lysate of ATRA (all-trans retinoic acid) differentiated neuroblastoma cells. In literature i've found that GAP43 protein is expressed during neuroblastoma differentiation. That's the problem: i try to load 30 micrograms of lysate in SDS-PAGE and a do a blot with anti-GAP43 1:200 and secondary HRP conjugated 1:5000 but i don't see any bands. is a problem of primary /secondary Ab concentration? or is a problem of mass of the protein? housekeepin' is OK, is not a problem of transfer!!!is anyone that can suggest me what can i do? is anyone that work with this antibody?
thanks!

---

1. 30 ug protein is enough for most western blot;
2. I am not familiar with anti-gap43. But I usually use 1:2000 dilution for primary antibody.
3. Do you have any positive control? I mean, some cell line that ABSOLUTELY express gap43. If it still does not wok, I suggest that something wrong be with your antibody. It happened several times in our lab.

---

Unfortunately i don’t have a positive control :( . I blot with another Ab to check neuronal differentiation and I find positive results. So I think that my cells are differentiated…Don’t you think that 1:2000 is too diluted? I think that GAP43 is a rare protein…Maybe I could blot the membrane with TBST-Milk 5% instead TBST-MILK10% or could use Ab diluted in TBST-MILK 0,5% instead of TBST-MILK 1%...Do you think it’s OK?

---

Hi.
I am in the midde of troubleshooting my Western blot as well...
Anyway, out of curiostiy, how long do you do your transfer for and at what voltage/power? Do you have SDS in your transfer buffer? I find that if I use SDS in my transfer buffer for one of my protein, there protein simply migrates right through the membrane. Also, what type of membrane do you use.
I have been discussing my problems with a few other reseachers and found that most people have a preferance for PVDF membrane...

Sindy

---

Dilution of primary antibody is very dependent on the antibody itself - some go up to 1:10,000 some as little as 1:100. You should check the literature from the company that you bought the antibody from - assuming that you bought the antibody. Or talk to someone in the lab who made the antibody. Typically I use PBST -5% milk for blocking. If there isn't much background on your blot then you can leave out the milk during the primary antibody incubation. Incubation could be anywhere from 4 hrs at rt to overnight in the fridge. Use PVDF membrane if possible, especially if your protein is less than 20-30 kDa. A positive control really would help you out a lot - even if you just clone the gene into a GFP vector or something - doesn't usually take much time and then at least you would be sure that the antibody was working.

---

I use a semi-dry transfert apparatus from Bio-Rad. We transfert 1h at 40 mA per gel (we can put up to 4 membranes). The voltage never go higher than 6-7 volts. And i've had proteins as low as 15 kDa stiking to the membrane.

As for the membrane, I use PVDF too. And yes, my transfert buffer contains SDS (with glycine, tris and methanol).

Hope this helps

---

Hi Syndy. I use fresh transfer buffer with 0.1% SDS.I set 150 mA constant for 2-2,5 hour. I use nitrocellulose membrane...I read that transfering to PVDF membrane are more difficult than to nitrocellulose membrane...if you have some trouble you can take a look at AbCam site. This is the linK:

http://www.abcam.com/index.html?pageconfig...e&rid=10413

---

But this is a protocol for low molecular weight protein...what could I do with a 43KDa protein? I transfer at 150 mA for 2 Hour with transfer buffer like your. I Blot with 5% milk and put primary Ab 1:200 in TBST-MILK 0.5%. i loaded 30 micrograms of lysate.. i saw a weak specific signal and much background...

---