Hi Everyone,
I am a bigginer in western blotting and I am trying to use it for validation of my proteomics data.
I have:
two regulated proteins with molecular mass of 40 and 73 kDa
one control protein 18 kDa,
and two phosphoproteins (with its sites validation) with molecular mass of 49 and 37 kDa.
Should I perform 5 separated gel and western blot set up to valid each protein separatly?
Or can I use one gel and western blot for validating 18, 40 and 73 kDa proteins and one for 37 and 49 kDa protein?
Thank you in advance
Dear Mahshid Zarrineh ,
The best idea is indeed to run everything in one blot. Just as you said, I would do two blots: one for the phosphoproteins and one for the non-phosphorylated ones (18, 40, 73 kDa). I would split the samples in two parts and run two gels simmultaneously. The same goes for the membrane transfer. Alternatively you can do first one gel, and freeze the rest of your sample to do the second blot another day.
Anyway, for the non-phosphorylated blot I would do as follows:
- I would slice my membranes into 3 regions: Below 30 kDa (to blot for 18 kDA marker), below 50 kDa (to blot for 40 kDa marker) and upper slice (to blot for 73 kDa marker).
- I would simultaneously incubate and blot my membranes with the different antibodies in different tubes. Doing this you avoid doing 5 blots. You also avoid having to strip (remove) the antibodies 5 times before incubation with the next antibody.
- After blocking your membranes with milk, I would blot with each primary antibody (at the correct concentration, usually 1:1000-1:5000) overnight at 4°C.
- Next day you wash with TBST, then you incubate the secondary antibodies for 2h at room temperature. Then you develop and scan, you can later put your membranes together to take a scan all together or you can join the images with some photo-editing software to reconstruct the whole membrane.
And for the phosphorilated blot (you can either do them simmulatenously or run this gel some other day):
- As your proteins are very close in molecular weight, cutting the membrane can be tricky. Therefore, for this one, you can just do one protein at a time: incubate with one antibody overnight, develop and take a scan.
- Then use any Stripping buffer to remove the primary and secondary antibodies from the membrane.
- Then re-block your membrane with milk, and after, your membrane is ready to do your other protein antibody incubation so you can take a second scan.
I hope this was helpful and good luck!
Best,
Julia
Depends on what you want to demonstrate. I'd always run the samples I need to compare on the same gel/blot. Comparing samples over several gels is difficult, I'd suggest to include references you can use for normalization on each gel and to do replicates.
Dear Mahshid Zarrineh ,
The best idea is indeed to run everything in one blot. Just as you said, I would do two blots: one for the phosphoproteins and one for the non-phosphorylated ones (18, 40, 73 kDa). I would split the samples in two parts and run two gels simmultaneously. The same goes for the membrane transfer. Alternatively you can do first one gel, and freeze the rest of your sample to do the second blot another day.
Anyway, for the non-phosphorylated blot I would do as follows:
- I would slice my membranes into 3 regions: Below 30 kDa (to blot for 18 kDA marker), below 50 kDa (to blot for 40 kDa marker) and upper slice (to blot for 73 kDa marker).
- I would simultaneously incubate and blot my membranes with the different antibodies in different tubes. Doing this you avoid doing 5 blots. You also avoid having to strip (remove) the antibodies 5 times before incubation with the next antibody.
- After blocking your membranes with milk, I would blot with each primary antibody (at the correct concentration, usually 1:1000-1:5000) overnight at 4°C.
- Next day you wash with TBST, then you incubate the secondary antibodies for 2h at room temperature. Then you develop and scan, you can later put your membranes together to take a scan all together or you can join the images with some photo-editing software to reconstruct the whole membrane.
And for the phosphorilated blot (you can either do them simmulatenously or run this gel some other day):
- As your proteins are very close in molecular weight, cutting the membrane can be tricky. Therefore, for this one, you can just do one protein at a time: incubate with one antibody overnight, develop and take a scan.
- Then use any Stripping buffer to remove the primary and secondary antibodies from the membrane.
- Then re-block your membrane with milk, and after, your membrane is ready to do your other protein antibody incubation so you can take a second scan.
I hope this was helpful and good luck!
Best,
Julia
Run two gels!
Normalize all your proteins bands based on your loading control protein (18 kDa) from the same membrane.
If your 39 and 47 kDa proteins are very close, run for longer duration. Alternatively, If these antibodies are monoclonal (hoping no multiple unspecific bands), you can add both (1˚Abs) antibodies together.
Pro tip- Stain more blots to have more confidence on your western result.
Don't discard blot. Stain duplicates instead.
-JP
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