Not much help, but how did you decide on your primary antibody dilution? I use 1:100 for some from SantaCruz.

I almost forgot, this is a membrane protein, correct? I look at membrane transporters and they require special conditions in the sample prep to avoid losing them. For example, 2% SDS in the lysis buffer and prolonged lysis to dissolve the membranes. Also, never boil membrane proteins. I add loading dye with beta mercaptoethanol and incubate at 37 C for 30 min. Boiling can aggregate membrane proteins.

Hi,

as we do in our lab, if we want to detect a protein has a high molecular weight, the transfer part should be 3 hours.

i hope that is help.

Hi Adam,

I decided on my dilution based on their website's recommendation. I've entertained the idea of increasing my concentration, but I felt there was something wrong with my prep, rather than the concentration of the antibody. I'm going to try the 2% SDS and I'll try your suggestion with the temperature as well. Thank you so much!

Most successful for me is ice cold lysis with that high SDS percent, which initially gives a very snotty thick liquid. The only way to make the consistency pipetteable is to sonicate with an ultrasonic probe in short 5 second pulses being sure to keep on ice the whole time and giving time to cool off between pulses. I've decided to stop pelleting and discarding debris and now I load directly from that sonicated lysate after a BCA protein assay. If done correctly you should get very little pellet if any even if you do decide to centrifuge. I recommend not pelleting at all though, because sometimes you're just pelleting precipitated SDS. Worst case scenario without pelleting is you get some stuff staying the well on the gel. In my experience this has no impact on my protein signal and size.

@Adam L Vanwert gave good tips on how to prepare cell lysates to analyse membrane proteins by western blotting. I will add on by reinforcing the importance of adding phosphatase inhibitors to the lysis buffer when looking for phosphorylated proteins. Also, I don't know which polyacrylamide concentration are your gels, but I will suggest you to run your samples on low concentration polycrylamide gels (e.g. 6-8%) and increase the time of transfer, since the protein of interest is quite large. Adding SDS to the transfer buffer (say 0.0375%) might assist protein blotting too.

Hope it helps!

Jonas, thank you for your comment. I always use SDS in my transfer buffer, but I have been going with the lowest concentration in the recommended range (I can't remember exactly the concentration). I go lower because more SDS can increase the current in the chamber, which, at least for me, will cause the BioRad power supply to lower the voltage to maintain a current at or below 400 mA. If I want to run it for 1 h at 100 V, it will sometimes be at 70 V by the end of the hour due to high current. Fresh buffer and low SDS helps to prevent this. Of course, overnight transfer at 20 V works well too, and then the current issue is avoided.

I have read that high SDS can prevent binding of the protein to the membrane, so that is another reason to not go too high I suppose.

Hi Adam,

I use 0.0375% SDS in the transfer buffer in a semi-dry blotting system from Bio-Rad, working at constant 23 V for 1-1.5 h and I have never experienced any problem. Moreover, I always use fresh transfer buffer.

I used a biorad semi-dry blotting system for 90 minutes on a constant 15V. Low % SDS-PAGE gel (7.5%) and larger pore size nitrocellulose (0.45 rather than 0.2) were also important.

For high weight proteins I used a high concentration of SDS (1%) in the transfer buffer and I transfered overnight at 40mA and in the morning 1h more at 100mA. All the transference was performed in cold and with a wet blotting system. I always used fresh buffe for high weight proteins.

I don't think the problem is the blotting technique. ErbB4 is a rare protein usually found only on neurons and then expressed at low levels. What cell type are you using? Why do you think you should be able to see it. A response to neuregulin doesn't mean that the receptor levels are high enough t see under the best conditions.

Very late reply! But I wanted to report back that I used @Adam L Vanwert's recommendations and it worked perfectly. I got exactly the effect we were hoping to see and it worked on all my tissue. I didn't put SDS in the transfer buffer, but the 2% SDS RIPA worked great. Thank you so much!!