Are you doing a ratio of pIRE1a / total IRE1a ? pIRE1 alone vs actin might not work, just like you don't see an induction of peif2a vs actin while peif2a / total eif2a is clearly increased. Also, which conditions are you using your antibodies and how do you prepare your samples ? phosphorylated proteins require specific precautions to not lose the phosphorylation during your WB.

Thank you Alexis. I use PhosStop and complete Mini with my RIPA buffer. And antibodies are working well since in another model it´s working ok (Hypoxia). You´re right with the total IRE1a, but since i saw the induction in hypoxia (with the same cells) i thought it would be a very strong induction. Also I thought that total IRE1a should not be modulated with the stimuli, but it´s true that eif2a should not change and it does.

I will take a look to total IRE1a antibodies.

Thank you very much

Hello Elsa,

Thank you for the answer. How do you prepare palmitate? and which dose? In order to clarify the duration influence I performed a time course from 2h to 27 hours... and the maximum level of phosphorylation reached is the same that the vehicle in the same time. But it´s true I ´don´t know total IRE1a. I´m sure that primary hepatocytes are very different to HEPG2 in order to react to palmitate...because i read a lot of papers with HEPG2 and I tried to reproduce the results in my primary hepatocytes...and nothing.

Thank you again for answering me.

Hi Bibiana, can you please check this article: http://www.ncbi.nlm.nih.gov/pubmed/19332540 .

From my experience, palmitate can be nicely dissolved in hot water. You may put the palmitate solute into DDH2O or Milli-Q water and slowly warm it up upto 65 to 70C. It will be clear solution. Then after couple of minutes bit cooling, you may add to your cell culture medium.

Also, try to use TBST buffer for phospho study  instead of  using PBST buffer. Best, Eunus

Sure? I read a lot of papers, and all the combinations are quite complicated. I did the easiest one... but now i doubt about filter the BSA coupled FFA. Because 0.22um is quite small and maybe a lot of BSA is detained and I don´t know really the amount of PA I´m putting to my cells. About the other topic, I already use TBST buffer for my WB. Thank you very much for your information and the article, althought most people work with hepatocyte line so I can´t wait the same in my experiments.

Bests regards

Endocrinology. 2011 Jun;152(6):2206-18. doi: 10.1210/en.2010-1369. Epub 2011 Apr 19.

Oleate blocks palmitate-induced abnormal lipid distribution, endoplasmic reticulum expansion and stress, and insulin resistance in skeletal muscle.

Peng G1, Li L, Liu Y, Pu J, Zhang S, Yu J, Zhao J, Liu P.

Fatty acid preparation and treatment

Sodium palmitate and sodium oleate were prepared according to a previously published method (27). Briefly, fatty acids were mixed with ethanol to a final concentration of 100 mm after which the mixture was repeatedly sonicated for 10-sec bursts at 200 W on ice until the mixture became a milky solution. Prepared fatty acid stocks were kept at 4 C and protected from light. Before use, the fatty acid was dissolved in 60 C preheated growth medium or buffer.

it works well, you can try, remember cite my paper when your publish your work,hohoh

Thank you very much for the answer Peng Gong. I´ll try it. I have a question about the protocol: what you kept at 4C is the milky solution? or it becomes clear after sonicating? Do you control the pH of the solution? and finally did you filter it? 

Thank you very much in advance, I´m quite worried about not preparing well my palmitate... and experiments are very laborious.

Of course if i used it I´ll cite your paper

:)

fatty acid preparing is quite difficult,  I kept milky solution at 4C, It is a stock. when it met  hot medium, it will be quickly dissolved. the most difficult step is soonication. you should have enough patience.

 no need to do other thing such as fliter, pH adjustment. if you can make milky solution, you get it. 

Peng Gong,

After 1 hour sonicating and vortexing with some heating (not more than 45ºC) I get the miky solution! it takes ling time to dissolve all the splodges!! Tomorrow I´ll make my experiments with HEPG2 and primary mouse hepatocytes.

I´ll explain here how it goes.

Thank you