transfer time

[nadia naeem]

what are the best transfer of different molecular weight protein time from gel to membrane? for 30 k dalton i gave 1hr and got result now results are not coming, ladder is clear but no band.

[admin]

Hello there Nadia,
usually I used to do fast transfers even in 30 minutes with good success.
What transfer conditions (voltage or current) are you using?


If you can see your ladder bands well (all bands including the small markers - ie 40 kda, 30 kda or less) then is highly likely that your transfer is going well.

If your ladder proteins are transferring well than more likely the rest of the proteins in the other lanes should be transferred as well.

If you see no ladder bands then you have a transfer problem.

How are you visualizing your bands (not the ladder ones) - HRP (horse radish peroxidase) after antibody incubations or coomasie?

Maybe you can upload a picture here of the gel so we could examine and help you troubleshoot?

[ethidiumbromide]

It sounds to me like you didn't load any protein into your wells. Did you change anything since the first time you did it?

Also, keep in mind that your ladder is also a positive control for transfer. If you see your ladder, but not your protein, then the problem is usually not the transfer.

[nadia naeem]

thanks for considering.

i use wet transfer with 200mA voltage, i can see the ladder but not clearly all bands.

i am visualizing bands after HRP conjugated antibody incubation and than visualising in gel dock system after 1 mins incubation in detecting reagent (Lumiglo and peroxidase 0.5ml both in 9ml deionized water).

i don't know how to upload pic here?

[nadia naeem]

I have loaded my protein, and i have increased transfer time, change antibody dilution. i can see the ladder but not very sharp bands.

[admin]

Hello Nadia,
thanks for responding.


All you have to do to upload pictures is click the box below (Go Advanced)

Then Click Button under "Attach Files": Manage Attchments

Then just Browse to the picture on your computer and submit your reply.

There can be several problems for you as you describe:

1) imaging-detection problem (check with someone elses results to see if your settings or the machine are working fine)

2) transfer problem as your ladder bands are not very sharp or strong

3) loading problem (you may be loosing samples or ladder during loading) this can be either human error or poor loading buffer. Make sure you use LOTS of sucrose to keep your samples from puffing in the lanes

4) developing problem - possibly your HRP is old or the bands are weak due to poor development conditions.

Please let us know what happened and upload a picture
cheers

[ethidiumbromide]

I would not recommend increasing your transfer time over 1hr. The proteins have a tendency to go through the membrane.

I'm still not convinced that you have your protein in the samples you are loading. Are these the same samples from the first batch you did where the transfer worked? Have you frozen and thawed these samples? How have you measured protein concentration? What concentration are you trying to load?

Can you describe your samples to me? What they were collected in? How were they stored? How were the concentrations measured? Also, it might help if you tell us what protein you are trying to visualize, as some proteins are very tricky to Western Blot anyway. Very acidic proteins do not transfer well, for example.

Let me know.

[nadia naeem]

My protein samples isolated from mesenchymal stem cells, stored at -20degreeC. i am visualizind CD90.
I am sure that i have protein in my sample because i am using stem cell protein sample which i have taken from heart and have done protein estimation by lowry's method it is 30ug/200ul it was the new sample not the one which i used earlier. i load 15ul sample in the well after dilutuing with sample diluting buffer in 1:1 ratio.

I am again doing western blot on wednesday can you give me some tips.

[nadia naeem]

Cardiac troponin I.jpg

GATA 4.jpg

Troponin I.jpg

these are the pic of my western blot

[admin]

Hello there,
thanks for posting your gels. It looks as though your transfer is not too bad (the bottom gel has a good transfer - the other 2 are tough to tell if its a loading/gel issue) but your gels could be improved i think.

I see several issues that can be improved:
1) Gel creation - are you using fresh buffers at the right pH? are you using a stacking gel?
2) Running of your proteins I can tell this as the bands could be sharper. Make sure you arent overheating your gels, and use/make fresh SDS buffer at the right pH.

You seem very close but the gel problems and running may be affecting your final outcome.

I would make sure you use fresh buffers for making your gels and make sure you mix them well after adding the polymerizing reagent. Let your gels sit until they harden well.
Are you using a stacking gel? If not I would add a stacking gel as well.

let us know how you go