We use wet blot transfer for our proteins the molecular weight of which is 18 -19 KDa. We use PVDF membrane for transfer and tris glycine SDS buffer contg methanol 20%. Our gel is 15% Stacking and 5% resolving gel 10 x 10.5 cm 1.5 mm thick.

The bands on the membrane are faint and no bands remain on the gel. We also get some additional high molecular weight bands on the gel which we suspect to be dimmer, but sometimes it is not visible clearly and sometimes we get good result. Is the transfer not efficient. We use alkaline phosphatase NBT and BCIP for detection.

Can anyone guide me on a good protocol for western blotting wet transfer method using NBT and BCIP. Also what condition shall I use for transfer since we do overnight transfers, can I can reduce the time

Also can I store by antibody used once and reuse it second time.

When you do the transfer you can try to change to Nitrocelulose membrane, some times it is better. Another posibility is to use a specific antibody instead of NBT and BCIP. The antibody way is much more sensitive. Are u using constant current for transfer? If yes how many mA ?
Write me more about the procedures and I will try to help you.

We use wet blot transfer for our proteins the molecular weight of which is 18 -19 KDa. We use PVDF membrane for transfer and tris glycine SDS buffer contg methanol 20%. Our gel is 15% Stacking and 5% resolving gel 10 x 10.5 cm 1.5 mm thick.
Run is 30 V, 160 mA, 30W, voltage constant, overnight run at 8 C
procedure followed after transfer
1. gel equilibrated transfer buffer 20 mins. PVDF membrane wet in methanol 1 min equilibrate in transfer buffer 10 min.
2 run overnight Bio-rad wet transfer
3.wash membrane with water
4. ponceau stg marking markers. destain.
5. blocking in 5%skim milk in PBS contg 0.02 %sodium azide11/2 hr
6 primary monoclonal Ab 2 hr
7. wash with PBS 3 x 10 mins then wash with Tris Nacl buffer 10 min
8. secondary Ab in tris NaCL buffer 1 hr
9. wash with Tris nacl buffer 4 x 10 mins
10. Development with Alkaline phosphatase contg NBT BCIP
The bands on the membrane are faint and no bands remain on the gel. Is the transfer not efficient. We use alkaline phosphatase NBT and BCIP for detection.

Can anyone guide me on a protocol for western blotting wet transfer method using NBT and BCIP. Also what condition shall I use for transfer since we do overnight transfers, can I can reduce the time

I would use a 30 min 100V (constant) for transfer. Then Rinse in TBS+tween 20 (0.05%) for a while. Block with 5% milk in TBS-T for 30 min and primary antibody over night at 4C on a rocker (slow speed). Wash 3x10 min in TBS-T and secondary antibody in 5% milk TBS-T for 1 hour at room temperature. Since your bands appear weak, maybe using an HRP conjugated secondary in combination with ECL. I use the one from Pierce and it works great. I wouldn't reuse the antibody, at least not until I got the method to work.

I would also try to use a much more sensetive WCL reagent. I.e. Pierce femto or Pico.
Both of them are very good to detec even a very faint signal.
Do a very quick exposoure.
Guy

I hope you have got the right solution by now, but do check the protein quantity you are loading go to >= 20ug and reduce the transfer time I keep for 200mM 1 to 1.5h only with icepacks at RT with biorad apparatus. or else you can do 2 step transfer 30min at 100mA and 1h at 200mA.
and MUST use 0.22micron membrane for small protein.
Gud Luck

hi,

Transfer of the proteins from gel to membrane is dependent on some factors like thickness of the gel,size of the protein,pore size of the membrane,voltage/amp for transfer, duration of the blotting.

Since as u mentioned ur getting some times and not getting some times.try to follow these conditions keep 1ug of protein and 70V,1hr with chilled buffer ( check out wht is the pore size of PVDF) after transfer try to check out blot with ponceau S stain.

Once u see your protein after ponceau,indicates there is no problem for transfer.

after that try to follow the conditions as u mentioned.

good luck.

Hello!
I ran western to detect APP (amyloid precursor protein), & it did not work as usual. Now I don't know if it's antibody problem or technical issue. I loaded 30 ug/lane protein, 4-20% gradient gel (Glycine-SDS-PAGE).
Electrophoresis at 160 V 1h and 15 min.
Transfer (Wet transfer): 100 V 60 min. (On ice)
Primary antibody: Overnight.
Secondary antibody: 2h
I really don't know where to begin with optimization. If you have any suggestions please let me know!!!

I am thankful!!!

I have done Westerns for 16 kDa protein (Hsp16) from C. elegans. I do a 1h wet transfer (Bio-Rad setup) at 100v/250mA in cold room onto NC membranes.
Blocking with 3% milk in TBS-T
primary for 1h at RT (depends upon the affinity of ab)
secondary (anti-rabbit-HRP from SIGMA at 1:10,000 dilution) for 45' at RT
3 washes with TBS-t between the steps.
Femto kit from Pierce for developing (15 second exposure)


I have used ALP-secondary and BCIP-NBT in the past using the same method in RBC and Plasmodium samples for ~25kDa proteins.

Is it possible that your proteins is expressed very low + your ab is not great. Does your secondary work with other primary ab. (add your secondary to a mix of BCIP+NBT...should turn dark brown in a few minutes).
Try using a new secondary ab (either ALP or HRP conjugate).

Good luck.