Dear Karen, I think it is not a big problem, and in trouble shooting steps, you can find the reason easily.

You did not get spot in WB. So you have not the protein, or you lost the protein or you had not suitable Ab.

You checked the Ab before, so it is not your problem (of course be sure about the same condition for checking the Ab and WB procedure for buffer type, buffer pH and ionic strength).

You are sure about protein expression.

So you lost the protein or the protein lost immunoreactivity.

Both lead us to unsuitable buffer for protein extraction.

My suggestion: prepare tissue homogenate (100 mg/ml in PB 100 mM pH 7.4  150 mM NaCl contains antiprotease cocktail.  Centrifuge at 4C and 12-14000 rpm for 10 min.

Some proteins sediment in SDS containing buffer, if you are not sure about that, replce SDS-PAGE by PAGE.

I predict and hope, you will have nice spot in WB.

Of course I a sure, you check enzyme activity and substrate in WB.

best

mehdi 

Hej! Thanks a lot for your answer. But I have used exactly the same buffer when lysing HEK cells and I was able to blot the protein... do you think think there could be a "tissue" specific buffer problem? 

Thanks,

Karen

Hi Karen, have you treated the tissue pellet with Laemmli buffer to see if your protein is in the pellet?

Also, do you add b-ME or DTT to your Laemmli buffer?

yes, exactly, I think you lost the protein in the pellet for unsuitable homogenizing buffer. I think phosphate buffer is better. But you have to change the buffer.

best

mehdi

Just add to Mehdi's answer, tissue culture experiments sometimes can't be used to represent in vivo experiments. 

HEK cells are kidney cells cultured in vitro and you trying are equating it to a protein expressed in mouse liver in vivo.

These are totally different assays. Things will be different.

Hej. Thanks a lot for all your answers. I have tried blotting the pelet once, but the blots were not the nicest and maybe I should redo this experiment to make sure I haven't lost any infromation there. 

Thanks again, I hope I can make it work now :)

Hi Karen, how are you homogenizing the liver? Dounce?, mechanical shredder?

If the pellet is very fatty, then you can could try TX-114 phase separation to get rid of some of the phospholipids.

Hey! I did the pelet again today and actually saw a band! wow, I am superhappy :D however, I did have some difficulties homogenizing the sample and didn't totally succeed. I shall absolutely try with TX-114, thanks a lot Steingrimur. Do you think it would also be possible to dissolve the pelets with Guanidinium or Urea? Or would you suggest percipitating the protein out of the pellets with TCA or so? And would it be worth to add some nuclease - I cenrifuged my samples extensively this time but nonetheless got this viscous structure, which I believe must be dna.

Thank you so much !!!

Hi Karen, good to hear that you found your protein!

Now comes the tough part to homogenize the pellet to extract the protein you are after.

First thing, don't use guanidinium because it will precipitate with SDS. TCA treatment will also precipitate phospholipids.

I would use a Dounce homogenizer to grind up the liver samples in an isotonic buffer, pH 7.5 containing 0.1% SDS and 4M urea. This step should also shear the DNA in your prep.

After you see that the tissue has been homogenized, pellet insoluble material using a low spin (~1000 rpm for ~1 min),

Collect the supernatant. If it is cloudy, you can try the TX-114 to get rid of lipids. If not, load it onto gels.

Let me know how it works!

Sorry, I should have said to use the Dounce with RIPA buffer containing 4M urea and proteinase inhibitors. 

Hej. I am aware of the Guanidinium SDS incompatibility, so usually I dialize the sample before loading but I guess your suggestion is even better :) 

thank you very much!

Hæ, if you have a viscous solution that looks like snot, then DNA is usually involved. 

Including DNA'ase will help, but I usually shear the DNA by repeatedly pulling and expelling the sample through a needle syringe.