Weird western

[A.A]

Hi Guys

It is an honour to be a member of this forum

I am struggling with my WB as I could not get it to work properly. I am trying two different antibodies to detect my protein. One of them is new and I have just started using it. The other one which I have been using for a while now gives a very good and a unique band however the band is much higher than the expected size. When I contacted the manufacturer, they suggested not boiling the sample as the protein is hydrophobic so when the sample is boiled they will for aggregates when cool down. Rather they suggested to heat the sample at 70 degrees for 5-10 minutes and leave to cool at RT (I don’t know if this makes sense, any suggestions are appreciated).

I did as they suggested but got no bands at all!!!!

As for the other antibody I got vertical lines from the top of the plot down to the expected size plus non specific bandsd (see attached images). Any ideas of what might be causing this??
To check that something might went wrong during running or incubations I probed the plot against B-Actin and got very nice bands see attachment

My protein is a plasma membrane protein however I take total protein. When lysing the cells I sonicate for three minutes (10 sec on and 10 sec off), the sample is sonicated while the tube on ice.

I appreciate any clues that might clarify what I am getting.

All the best

[admin]

Hello there, thanks for joining the site and welcome!

The first blot above seems like it has an SDS-page gel problem, but if the lower blot is a stripping of the top one, then you may not have an Sds-page gel issue. Also I see 3 marks there, but would be nice to see a ladder to see how the bands look - would clarify if it is a gel issue or not. Could also be a loading buffer issue (is it prepared fresh with beta-mercaptoethanol? ie is it really smelly? Do you add the same to your ladder?) Also if you have a positive control, of your protein that would be great to see run with your samples as your samples may have issues - salt, weird contaminants etc. causing the gel problems/blot issue...
Let us know how you got the bottom gel is it strip and reprobe?

Also, I would try to heat your samples normally (ie 90 degrees at least for a few minutes) and compare the two antibodies. Also, try to strip and reprobe the same gel with your actin and another control to see how your gel is running.

Let us know how your progress goes and best luck on your experiments we are here to help.
cheers

[A.A]

Thank you for the response

The actin blot is of the same membrane without even striping however the exposure time is much less. In the actin it was for 5mins whereas for the other antibody it was more than half an hour. Additionally, I did the actin as the second probe for about 16 hours which may reduced the signal of the first one (the first blot).
The marker was fine however; I didn’t use the same loading buffer. I think it is unlikely to be the loading buffer issue as this should effect the actin as well.

Do you think having excess of protein can cause this. I loaded about 60ug of total protein?

OR too long sonication time ? (remember, it is a membrane protein)

[Anastasia_S]

Your WB is very strange!
I do westerns for membrane protein also and I'm sure sonication does not affect how well it runs (I sonicated for 1,2,3 min - I don't stick to protocol precisely ; also, sometimes I resuspend the sample by sonication after I defrosted it)
When I have gel overload, I gel really thick bands (like you actin, actually) but they are normal bands; well, sometimes one side is a wee bit thicker than the other
I had the band turning patchy once (it wasn't as bad thou) and I think it's because I didnt mix up the protein pellet with SDS loading buffer well. But I would think the actin should be affected also if it is the case.

I would say it is either the SDS_PAGE problem (but inly in the first third of the gel, and then it ran properly). Or, which is more likely, it is the antibody problem.

With respect to your "good" Ab, is it likely that you may have glycosylation on your membrane protein and thus higher MW?

[Tfal]

What type of membrane protein? What are you doing for to get "total protein"? If you are looking for intrinsic or transmembrane protein and are using the same fraction as for B-actin or you are only doing lysis followed by removal of cell debris (e.g. 15-20 min 17 000g centrifugation) without resuspension you might be discarding much of your protein. Or your protein might not be sufficiently abundant and you might need some purification (narrower fraction by salting e.g. (NH4)2SO4 precipitation).

good luck