I would like to measure levels of different phospho-glucocorticoid receptor forms in rat brain tissues using Western blot. Could someone recommend me any reliable antibody and probably special conditions/protocol how to extract the protein without loosing and avoiding any loss of phospho-GR isoforms?
Marcella, I've been out of town and just noticed your question concerning NMDA receptor expression changes following stress, which I wanted to follow up on. No, we have not done that experiment, yet, but have been working on chronic stress changes in glutamate and GABA circuits in the PVN, which we will submit for publication soon. In short, we find an increase in glutamatergic and a decrease in GABAergic innervation of PVN neurons with chronic variable stress.
Hi, I used anti-phospho-GR antibodies in human cells (A549). In my case I used p-Ser211-GR (Cell Signaling Technology) and p-Ser203-GR (ab79268, Abcam), and they worked fine. Remember to compare the signals with total-GR to have a good interpretation of the appearance of phospho-GR isoforms. For the protocol, was really important to add phosphatase inhibitors to the lysis buffer and to the electrophoresis and transfer buffers. Hope this help although I worked with human cells. Good luck!
thanks Pedro. Well, I am working with rat tissues but anyway, those antibodies should work. Do they provide anyunspecific bands as well? Please, could u send me some more detail about using phosphatase inhibitors? how much should I add esp. into electrophoresis and transfer buffer?Does it not interfere with the whole procedures? Please, I would appreciate you advices. Thanks.
I added 5mM NaF and 2mM Na3VO4 (both from Sigma) to the PBS for washing the cells, also to the lysis buffer (Tris:HCl 50mM pH7.4, NaCl 150mM, EDTA 1mM, 1%Triton-X100, 5mM NaF, 2mM Na3VO4, Aprotinin 2μg/ml, Leupeptin 2μg/ml, PMSF 100μg/ml, Iodocetamide 20mM), and the same concentration to the electrophoresis and transfer buffer. In my case was not affecting the whole protocol. Remember to transfer during enough time for this 95kDa protein, and not too less or too much, otherwise the GR will remain in the gel or transferred beyond the membrane (in my case I used 0.45μm Bio-Rad (cat#162-0115) membranes and a constant voltage of 100V during 120 min).
Regarding the unspecific bands, yes, appeared some unspecific bands but I can remember they appear low in the gel, so was easy to distinguish them from the GR by obvious differences in size. In my case was also clear because we made nuclear and cytoplasmic extracts: in resting conditions total GR were in the cytoplasm and we could not detect p-Ser211-GR band, and when cells were activated with dexamethasone we could see the total GR disappearing from the cytoplasm and appearing in the nucleus, and p-Sr211-GR appearing mainly in the nucleus. If you use total whole cell lysate, you will not see p-211-GR or p-203-GR in resting conditions, and they will appear after 10nM dex treatment during 2h. Hope this helps!
excellent. Hmm, actually I wanted to run phosphoGR in whole cell lysates because of small brain tissue, from unstressed and stressed rats, so I wonder whether I can still get some basal signal. I am using Qproteome Mammalian Protein Pre Kit, which should extract nuclear proteins as well. Since the phosphoGR and GR on its own are very sensitive for degradation and using frozen tissue I have to defrost it and then homogenize useing the pestle, I am affraid I can loose the signal. Do u think I can still get the signal using the whole tissue extract from controls or not at all?
Thank u a lot for your advice, I really appreciate your fast response :-)
By the way, it seem that ab79268, Abcam antibody is not available anymore. thats sucks. Any idea how to get at least a small portion?
Sorry, I have no experience about whole tissue extract, and regarding the Ab, if it's out of the catalogue, I would suggest to ask Abcam if they can recommend you other source or other Ab. I'm sorry but I cannot help you with this...
thats fine, anyway, thank you once again for your help and let we see what I will get :-)
The Cidlowski lab has developed phospho-specific GR antibodies. Look up JR Cidlowski (e.g., Galliher-Beckley et al., Mol Cell Biol. 2011 Dec;31(23):4663-75; http://www.ncbi.nlm.nih.gov/pubmed/?term=cidlowski%2C+glucocorticoid+receptor%2C+phosphorylation).
yes, but they wont provide the antibodies for testing unless included into collaboration, at least that was the answear when we asked for GR translational isoforms antibodies :-) but anyway, thank you.
Thats fine, we have to work with what is available at the moment :-)
by the way, Jeffey, I see u r working a lot with PVN and synaptic plasticity. One question, have u ever tried to measure NMDA receptor expression in PVN following stress? If so, what can it say, what functional significance might it have for PVN physiology?
Pedro, one more question. Do you usually add phosphatase inhibitors also to blocking buffer? Do you use 5%BSA in TBST?
Hi Marcela, we were using the Odyssey-LiCor system to analyze the WB membranes, so we were using Odyssey Blocking Buffer (Li-Cor Biosciences, cat#927-40000) and didn't add phosphatase inhibitors to it. I'm looking for a paper that I read in the past showing some experiments running the electrophoresis and the transfer with and without phosphatase inhibitors and the corresponding results detecting a phosphorylated protein, and showed some improvement when adding the phosphatase inhibitors to the buffers. I cannot remember anything about the blocking buffer. I'll send you the paper if I can find it. Anyway, what I'm totally sure because I tested it several times in parallel, is that you have to add the phosphatase inhibitors to the lysis buffer, this was determinant for me to detect phospho-GR isoforms by WB. I added to the electrophoresis and transfer buffers because the paper, but never did comparison of both protocols in parallel, I did only for the lysis buffer. I will find the paper. Good luck!
Thank you a lot Pedro. Well, let we see whether I got something tomorrow, I am running few samples from stressed animals, so I am curious. Its always better to work with treated cells, at least you know what to expect :-) Anyway, thank you for your help and "read" you soon.
I've had good experience on WB for ph GR S203 and ph GR S211 antibodies on both murine and human cell lysate samples.
phosphorylated murine glucocorticoid receptor S220 (Cell applications, cat nr CC1038) and S212 (Abcam, cat nr ab79268).
The ph GR S226 from Abcam, did not perform well.
Also see my fresh out article in Eur J Pharm :
Mitogen- and stress-activated protein kinase 1 MSK1 regulates glucocorticoid response element promoter activity in a glucocorticoid concentration-dependent manner.
Beck IM, Clarisse D, Bougarne N, Okret S, Haegeman G, De Bosscher K.
Eur J Pharmacol. 2013 Jul 4. doi:pii: S0014-2999(13)00501-3. 10.1016/j.ejphar.2013.06.032. [Epub ahead of print]
Good luck
Ilse
Thanks a lot Ilse, unfortunately ab 79268 is no longer available, so cannot order it anymore. regarding pGR211, there is also good one from Cell signalling. However, I would like to ask you, do u have also some unspecific bands when using mouse lysates and pGR211???
The cell signalling one you are referring to, was also recommended to us by Michael Garabedian.
As for the aspecific bands, I had to recheck the full blots.
The blot was pretty clean. Specific signal for ph GR S211 and on one blot I found a low signal additional band at about 60 kDa
what about unspecific bands above 100kDa? I found kind of strong signal above 250 kDa. See enclosed image. These are rat brain samples, calibration curve. Dilution 1:500. I wonder whether these unspecific bands could be a consequence of a high antibody concentration or something else. I used 20, 30, 40 and 50 ug of protein for the first 4 samples. I would appreciate if you could share your picture with me, to get better idea. And yes, Michael also recommended Cell Signaling antibody to me, that was the reason why I bought it. It is just pitty ab79268 is not selled anymore. Currently I am testing another abcam antibodies against pGR Ser 226 on rat tissues, so, let we so. However, by now I would at least like to run pGR211 since I got some signal. Thank your for sharing your experience :-)
Marcella, I've been out of town and just noticed your question concerning NMDA receptor expression changes following stress, which I wanted to follow up on. No, we have not done that experiment, yet, but have been working on chronic stress changes in glutamate and GABA circuits in the PVN, which we will submit for publication soon. In short, we find an increase in glutamatergic and a decrease in GABAergic innervation of PVN neurons with chronic variable stress.
Marcella,
We did not observe aspecic bands above 100 kDa
Perhaps you can contact Abcam to ask about the reason for discontinuing the Ab... may be a temporary suspension ?
Best of luck to you
Thank you a lot Ilse. Well, it seems they are not going to sell this antibody anymore, I called them, but did not get answer why. So, as I understand, the image u attached was done with Cell Signalling pGR Ser211 #4161 antibody, right? Did u use mouse tissues< how much protein did u load? I am using 4-15% gels, so I would not expect some protein will remain at the top of the gel.
Hi Marcela,
Might be a misunderstanding
As I indicated before: our phGR S211 (i.e. phosphorylated murine glucocorticoid receptor S220) was done with an antibody of : Cell applications, cat nr CC1038
The lysates were made from murine cell cultures of a fibroblast cell line.
Lysis was performed using our quick lysate protocol (see below) and then we load 20-25 µl. Loading controls are performed via post-hoc checks of a constitutively expressed protein such as tubulin, actin, ...
Gel percentage was 8%
Blocking was performed in 5%milk in PBS
and antibody incubation was performed in 5%milk in PBS + 0,1%Tween, 1st Ab dilution: 1/1000
Ilse
QUICK lysis in 1x SDS sample buffer
Composition 1X SDS sample buffer (as in TNT protocol of Promega)
50 mM Tris pH6,8
2% SDS
10% glycerol
bromofenol blue
100 mM DTT (add fresh)
• Prepare 1X SDS sample buffer -- + DTT 1/10 (add fresh)
• work on ice
• Rince cells with (about) 1 ml of PBS (gently)
• Suck dry
• Add 150-300 µl of 1XSDS sample buffer (depending on the confluency of the cells)
• make slimy ‘boogers’
• Sonicate sample, 1’
• Boil for 5min at 95°C
• (Store at -20°C)
Process for WB
I got it. thanks a lot. Perhaps it would be worth to try your antibody. But its questionable whether we will get the same "clean" signal" with rat tissues. Anyway, thank u a lot for your advices :-) have to think about what to do.
Hello Marcela.. I am in this exact moment performing western blot to evaluate P-GR (Ser211) in rat muscles. I let for overnight right now incubating the primary Ac. I'm trying Bioss http://biossusa.com/store/bs-3168r.html because it was the only one I found with reactivity to rat. I've seen almost everone using Abcam nicely, but always in human or mouse samples, that is not for my samples. Well, if my western (performing now) works fine I'll tell you ok?
great, that would be very useful. there is one antibody from Cell Signaling against Ser211 which worked for ras as well. However, with certain tissue, it gave 2 bands, but there was still one corresponding to actual molecular weight. let me know how about your antibody and westerns, if possible could you also attach the picture? Thanks you and good luck.
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