9 replies to this topic

[asta6801]

Hi there

I have an issue with my blots. I had been getting good results using these lysates, but now I am not getting any bands coming up for the proteins I am probing for, and only some bands coming up for beta actin (some are faint, some are normal). I have attached a picture of the most recent blot.  People in my lab have suggested that the membrane may not be getting soaked in the antibody properly. Does anyone have any suggestions, or has seen something like this before?

Thanks  2013.02.21 low res.pdf   17.08K   80 downloads

[bob1]

A few more details about your gel and transfer and developing process would be good - otherwise the potential problems run into the 10's

[asta6801]

Thanks, I can provide more details but hopefully this is enough:

These were run on a 10% gel at 80V for 1.5 hours. The transfer was done overnight at 4 degrees at 30V onto a PDVF membrane. To visualise the gels I washed 4x 5 mins with TBST after the secondary antibody, added Luminata Forte and put the gels into our BioRad Chemidoc and exposed for 2-5 mins.

[bob1]

How do you do your antibody incubation (shaker?, tube roller? volume of Ab dilution?)?

I assume you loaded equal amounts of protein in each lane?

How did you set up the transfer - rolled out the bubbles?  Ensured that the pads etc were fully wet and no bubbles?

[almost a doctor]

Did you stain your membrane after transfer?  It looks to me either the proteins haven't transferred properly, or as your labmates suggest, it's not being properly incubated.

[asta6801]

I did the antibody incubation again, to see if it was to do with the membrane not being properly covered by the antibody/milk solution, but unfortunately got the same results as before. Looks like it may have been the transfer. I usually soak the membranes in methanol for around 1 min, and soak everything else in transfer buffer. I do check to see that there are no bubbles, and roll any that are there out. Perhaps this time I just wasn't thorough enough about it. I'm not able to check this though since we have run out of ponceau stain, unless anyone has another way of doing it?

Thanks all for your help, hopefully I will have better luck next time.

[bob1]

You can coomassie the gel to see if there is residual protein - kind of the inverse of doing the ponceau.  It doesn't really tell you if the protein is on the membrane though, only that it has migrated out of the gel.

You can also coomassie stain membranes, but it damages the protein so that  you can't probe with antibodies afterwards.

[mdfenko]

after soaking the membrane in methanol, do you then soak in buffer (you should)?

are the lysates the same exact samples that worked before? they may not be good anymore.

[asta6801]

Thanks, I might try comassie the membrane then, I don't need to use it so doesn't matter if it doesn't work.

I do soak the membrane in buffer after methanol. Some of the lysates were new ones, some were older that I use as positive/negative controls. Generally I find that they are good for many months if kept in a -80 freezer, and they were the ones that appeared to work the best when I probed for beta actin.

[mdfenko]

don't coomassie stain the membrane after blocking (and further processing). you'll end up with a totally blue membrane. you can only stain the membrane before processing.

what is the size of your protein of interest? what is the pore size of your membrane?

have you tried transfer with a membrane backing up the membrane to determine if you are getting blow through?