13 replies to this topic

[mickey]

I am a Newbie to research, and undergoing molecular biology study. I got problems with weak Signal in western blotting.

I need to undertake a lot of protein detections. At first, I use 4%-12% Nupage from Invitrogen, the band is great.

I detect some common protein in neuroscience study like PS-1 and APP series.

After about 3 times of using these commercial GEL, I began to cast sds-gel of Tris-glycine system my self.

I use similar electronic and antibody merit when I run Nupage and self-cast gel like Electrophresis:100v voltage consistant when I running 15% acrylamide/bis gel, the currency stays 22-26mA at stacking part and 12mA seperating part the process lasts about 140min. I always incubate my memebrane at 4 degree over night about 12 hours, and wash with 0.2% tween TBST before and after secondary antibody incubation 20minx3times.

The electronic bloting I use bio-rad minigel system with 55V voltage consistant and got 150-200mA in CAPS(pH11 with NaOH) bufer with 10% methanol for 85 min.

I carefully equilibrate the sds gel for 15 min before blotting also equilibrate membrane in methanol-MillQ-transfer buffer when I use PVDF. All my bands are developed with ECL reagent.

But the problem is weak signal, I tried 10%,12% and 15% gel several times, although I got better signal when I use gel with less density. But I need 15% to seperate a lot of proteins around 10KDa. When I use 15% gel to transfer the seeblue plus2 marker sometimes couldn't be fully transferred with miss of 250kDa and 148kDa. I use 100V consistant 120min for transfer once but the result is just a little bit better.

Thank you so much to read a problem so long. And I am waiting for your great opinions to help solve my problems.

[Prep!]

i think ur transfer efficiency is weak... have u ever stained the transferred gel and seen if all the band has been transfered? if no then do tat and addition of 0.01-0.05% SDS to the transfer buffer shud do the trick.
if transfer is proper, then check the antibody dilutions and incubation times... not forgetting wash systems and blockng!!!
Best luck!!

[mickey]

I will undertake a gel staining.I knew the SDS stuff, but how does it work? I knew that methanol working for detaching protein from SDS, then how about the SDS's role in transfer?

I read a paper about reuse transfer buffer at least 7 times to maintain relatively good results(Transfer Buffer Containing Methanol Can Be Reused Multiple Times in Protein Electrotransfer Colin J. Pettegrew,* Renuka Jayini,* and M. Rafiq Islam). They even use the same buffer as SDS running buffer with 0.1% SDS.

I am totally confused. Sorry for so many questions.

[mdfenko]

the methanol will strip the sds from the protein but adding some to the buffer ensures that the protein will have enough sds bound for long enough to elute from the gel.

[mickey]

I stained the gel after transfer with Bio-Safe Coomassie Stain , I got clean blue bands on both 12% and 15% polyacrylamide gels. Although not so strong, but it is really bothering me so much. Thank you guys for suggestions. I will try to improve my protocol.

By the way, when I use ECL for detection, sometimes there was signal on film like flame, is it the resisted detection solution? How could I improve my operation? Thank you so much and sorry about so many questions.

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[Prep!]

best luck with the protocol.. do come back if the problem is solved (even if its not!!!   )... and i have not worked with ECL but normally such apprerance is due to over incubation with substrate or improper blocking... over heating of thew western.. i hope u using ice!!!
i was also wondering why wud u use a pH 11 buffer for transfer??!!! is the pI of ur protein too high say sumwere near 9 or so??!!! if not then the normal towbin buffer shud be fine.. cause may be at this high pH ur protein is getting degraded or sumthing!! (just a thought!!)

Edited by Prep!, 26 July 2010 - 08:32 PM.

[mickey]

Prep!, on 26 July 2010 - 08:28 PM, said:

best luck with the protocol.. do come back if the problem is solved (even if its not!!!   )... and i have not worked with ECL but normally such apprerance is due to over incubation with substrate or improper blocking... over heating of thew western.. i hope u using ice!!!
i was also wondering why wud u use a pH 11 buffer for transfer??!!! is the pI of ur protein too high say sumwere near 9 or so??!!! if not then the normal towbin buffer shud be fine.. cause may be at this high pH ur protein is getting degraded or sumthing!! (just a thought!!)

Some people use CAPS with PVDF for sequencing also CAPS only work between 9.5-11 pH. So I　undertook this protocol. I use ECL on ice also I pre-open air conditioner before I use the film developer in dark room. It's wired because sometimes it occurs sometimes not.

[Prep!]

yeah but if the pI of your protein is not so high then i would use the conventional buffer than such a high pH. and by ice i meant the run should be in ice!!

[mickey]

Prep!, on 26 July 2010 - 10:45 PM, said:

yeah but if the pI of your protein is not so high then i would use the conventional buffer than such a high pH. and by ice i meant the run should be in ice!!

Thank you for the rapid reply. I use wet transfer tank equipted with ice block. I will try Tris/Glycine transfer this time. Thank you

[mdfenko]

transfer with caps is fine. you may want to determine if you are transferring too long, at the higher pH the proteins may be moving faster in the electric field.

what is the pore size of the pvdf and the size of the proteins of interest?

if your proteins are small then you may want to use a pore size of 0.22 um or smaller. if you are using 0.45 um then you may want to try a backup membrane to ensure that you are not blowing through (also, stain the gel to see if any is left behind).

[mickey]

mdfenko, on 27 July 2010 - 05:58 AM, said:

transfer with caps is fine. you may want to determine if you are transferring too long, at the higher pH the proteins may be moving faster in the electric field.

what is the pore size of the pvdf and the size of the proteins of interest?

if your proteins are small then you may want to use a pore size of 0.22 um or smaller. if you are using 0.45 um then you may want to try a backup membrane to ensure that you are not blowing through (also, stain the gel to see if any is left behind).

Thank you so much. I use 0.22um PVDF or nitrocellulose and my protein size ranged from 9kDa to 55kDa. The problem is when I stain the gel with commassie blue from Biorad, there are definitely bands on the gel after transfer.

The western is really puzzled, I tried same samples when I use NuPage from Invitrogen, the signal is more than 10 times different.

[mdfenko]

mickey, on 29 July 2010 - 05:13 PM, said:

Thank you so much. I use 0.22um PVDF or nitrocellulose and my protein size ranged from 9kDa to 55kDa. The problem is when I stain the gel with coomassie blue from Biorad, there are definitely bands on the gel after transfer.

so you are not getting efficient transfer? are all the bands still in the gel (including the 9kDa)? (i would think that the 9 kDa protein would pass out of the gel rapidly and may pass through the membrane)

did you equilibrate the gel with transfer buffer before assembling the transfer sandwich (not always necessary but if you are not successful without it then you may want to try or vice-versa)?

did you ensure that there is full contact between the gel and membrane?

[mickey]

mdfenko, on 30 July 2010 - 12:24 PM, said:

mickey, on 29 July 2010 - 05:13 PM, said:

Thank you so much. I use 0.22um PVDF or nitrocellulose and my protein size ranged from 9kDa to 55kDa. The problem is when I stain the gel with coomassie blue from Biorad, there are definitely bands on the gel after transfer.

so you are not getting efficient transfer? are all the bands still in the gel (including the 9kDa)? (i would think that the 9 kDa protein would pass out of the gel rapidly and may pass through the membrane)

did you equilibrate the gel with transfer buffer before assembling the transfer sandwich (not always necessary but if you are not successful without it then you may want to try or vice-versa)?

did you ensure that there is full contact between the gel and membrane?

Thank you. Only bigger than 30kDa stained on the gel. I alwasys equilibrate the gel for 15 to 30 minutes. Also I cutted a 10ml pipet to get rid all the bubbles.

Now I could get good signal from 10% and 12% gel, also bands I want from 15% gel with little background. Problems are I now do some overexpression the signal seems not so good on 15% gel. Although other people's data are from NuPage or Tricine system, just because I want 9kDa and 70kDa on the same PAGE.

[mdfenko]

mickey, on 30 July 2010 - 05:37 PM, said:

Thank you. Only bigger than 30kDa stained on the gel. I always equilibrate the gel for 15 to 30 minutes. Also I cut a 10ml pipet to get rid all the bubbles.

Now I could get good signal from 10% and 12% gel, also bands I want from 15% gel with little background. Problems are I now do some overexpression the signal seems not so good on 15% gel. Although other people's data are from NuPage or Tricine system, just because I want 9kDa and 70kDa on the same PAGE.

you may want to try a gradient gel to visualize 9 and 70 kDa on the same blot.