Hi Francesco,

we need some precisions to help you

I have few questions:

What is the % of your gel?

how long do you do the transfer on the membrane,  what is the voltage and what is the composition of you buffer and the pH?

what is the concentration of your protein?

Best

Celimene

Hi Francesco, 

Actually, there is different factors that can influence your western blot results such as gel concentration, migration and transfer quality. Furthermore, it might be tricky if you use antbodies and substrates of bad quality. 

Please try to specify whis step affects your final results. 

Good Luck. 

Best

Hamadi

What running buffer do you use? MOPS based running buffer resolves high MW bands significantly better than e. g. MES running buffer.

Dear all thank you very much for yours answers; 

Now I'll try to answer to your questions to have a better feed back:

@Celimene : I have tried different % of gel ( 8%- 6%- 4-8% gradient) and no one has enough power resolution for my purpose. I have transferred ON or 1 hour.  The "problem" deals with the resolution power of the gels. I don't have problem in protein detection, but now I want to perform a more deep analysis of my protein of interesest. I used buffer based on Tris-Glycine buffer ( + SDS during the running or + MetOH during the transfer; but however is not a problem of transfer or protein concentration).

@Hamadi: It is not a problem of antibody ( it is a tag protein ) and I can perfectly detect the single band; but I know that this band represent two different forms of the same protein and I want to separate them.

@Cristoph: The running buffer is a Tris-Glycine + sds 0.1% ( and I have always used this buffer for all). Do you known same method-papers that show results with MOPS buffer compare to other buffers?

Thank you very much

Francesco

Hi Francesco,

we use the NuPAGE system from invitrogen. The manual (https://www.lifetechnologies.com/content/dam/LifeTech/migration/en/filelibrary/protein-expression/pdfs.par.1058.file.dat/we22841-nupage-faq-fhr.pdf) contains migration patterns on the last page. We are using 4-12% Bis-Tris gels with MOPS running buffer to routinely resolve high MW proteins (~ 100 kDa). Also, there is some additional information here: http://openwetware.org/wiki/Sauer:bis-Tris_SDS-PAGE,_the_very_best

all the best,

Christoph

Have you tried 10% and let the proteins migrate more than usual till you loose protein and see better separation of your markers near ~100kD

Try to use 4to 12% gel. Run at low voltage.

How close is the expected size of the two bands you are trying to resolve? 

I've looked at high molecular weight proteins (>150 kDa) on 7% acrylamide gels and you can differentiate protein bands that are glycosylated (for example) from the unglycosylated form.

Are you looking at something like a lipid modification or a smaller cleaved form?

I can't add much to what people said above, except to pour gels with long a long stacking portion. For example, if my gel is 6 cm from the bottom of the wells to the bottom of the gel, I will mark the glass 2 cm from the bottom of wells and pour my resolving gel up to that point. This means a third of the entire gel is resolving - I get very crisp bands this way. 

Hello,

There many factors that can affect the resolution of your western blot:

- You can decrease the percentage of Acrylamide in your gel to make wider pores.

- let your running be at a lower voltage for a longer period of time in order to get a good and enough separation of your bands.

- you can increase the wash duration or increase the percent of tween in your wash buffer to get clearer image without any noise between the two bands you need.

Best.

Dear Francesco, I agree with Christoph. MOPS running buffer can really improve your discrimination power. Both discontinuous and gradient gel will suit, only make sure to run the gel for a suitable anout of ime and do not run in too fast. We routinely work with TrkA bands with high molecular weight.

Best

Francesco

Hi, generally speaking,antibody is major cause of WB failture,but it seems that it is not a problem in your work. If you want to separate two different bands around 100KD, I suggest that you can increase the dilution of the second antibody in order to exclude non-specific affinity .And washing throughly with TBST after incubating second antibody.