SOLVED:
Thank you to everyone who helped! Although it turns out that the issue lay in the plants, I have learned a lot about how others handle proteins and have improved my overall technique. After much testing, it turns out that eIF2A is MUCH more strongly phosphorylated in un-expanded leaves, and the older the leaf is, the less phosphorylated eIF2A is present. As I try to use mature leaves in my experiment, this has caused the difficulties described below. It turns out that my western blot was working perfectly - there really was a difference in the amount of phosphorlyated eIF2A present in my samples.
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Using an eIF2A-P specific antibody on total plant protein extracted from Round-up (glyphosate) treated N. benthamiana, only some of my lanes show the presence of the eIF2A protein. All samples should contain the protein as glyphosate has been shown to trigger eIF2A phosphorylation via GCN2. Positive control working (data not shown).
Roundup is working. 150umol Roundup is sufficient to cause eIF2A phosphorylation in A. thaliana. Gel is loaded carefully with no spilling. SDS-PAGE running smoothly. Transfer is working. Antibodies are binding correctly. I suspect user error, not issues with materials due to the perfectly alternating bands. Bands in other gels also alternate. Any suggestions?
IMAGE: eIF2A-P shows at 37kDa, the band at 50kDa is Rubisco. All lanes contain equal amounts of protein.
METHOD: Total plant protein extracted using equal volume buffer (20mM HEPES, 0.1M KCl, 10% glycerol, 5mM MgCl2, 5mM MgSO4, with 10µL/mL of Sigma Protease Inhibitor Cocktail and 10µL/mL β-mercaptoethanol added directly before use) to plant tissue, ground mechanically, then centrifuged to remove all cell debris.
Plant protein measured via Qubit fluorometer, 10ug protein loaded into an Eppendorf test tube and the total volume made up to 22µL with SDS loading buffer (1% SDS, 10mM Tris-HCl (pH 6.8), 1mM EDTA, 80mM DTT). To this 3µL of loading dye (0.5mg/mL bromphenol blue in glycerol) was added. Samples heated at 85'C for 5 mins, loaded into 10% Acrylamide 2% SDS etc gel, run, transferred onto 0.45µm PVDF Millipore Immobilon-P Membrane in transfer buffer ( 20% methanol, 192mM glycine, 25mM TRIS-HCl (pH 8.3)) at 90A for 16 hours. Stained with Ponceau S.
Antibodies working well, so I won't describe that. Chemiluminescent exposure.
Do you mean this is a phophoprotein blott? but why do not use phosphatase inhibitors? i am not an expert of plans, anyway. can you blott the elf... total protein too?
decrease the amount of Primary Ab, also use phosphatase inhibitor in sample preparation
Previously (when working with A. thaliana) there hadn't been a need for phosphatase inhibitors, but now it seems it might be a good idea. Any suggestions on brands?
hm. i repeat i am not a plant expert. however, i figure out that elf2a is the plant analog of p53. if i am right. In this case it goes into the nucleus due to your round up. And sticks to DNA so i suppose it is harder to sample. Rubisco stays in its place, it looks easier to bring into sample. So i think you have at least two effects causing variability (for elf2aP): phosphatases and dna-binding. You should check elf2a content of the samples which works against you. And the last: theoretically, Ser-P or Thr-P easily degradate at higher temp (this is pure chemistry). For SDS-sample prep, lower the temp below 50 deg C.
when ever looking for phospho proteins, you must protect them and carry out all protein work on ice. I use sodium pervanadate or phos-stop by roche. You should consider probing your blot with total protein for EIF2 to confirm it is there. my only explanation for alternate signals on your blot is that there is insufficient protein loaded into those wells
If you hadn't needed phosphatase inhibitors before, the answer could be either sample degradation or low elf2a:total protein ratio. It's an extra step and added to an already long process, but you may need an isolation step for your target protein.
Hi Kate,
I worked with eIF2alpha in Toxoplasma and I always added phosphatase inhibitors (NaF and beta-glycerophosphate since it is phosphorylated on a serine residue) when I prepared my protein lysate. In addition, I also checked for total eIF2a since protein translation is down regulated when eIf2a is phosphorylated. And in some cases I saw a reduction in the amount of total eIF2a after some cellular stresses. Another control I usually applied when I had weird results was boiling the samples directly in reducing SDS-sample buffer to be sure that dephosphorylation does not occur. The downside is you need eIF2 total to normalize the samples. Hope this helps. Good luck.
I would also check for potential problems with cell lysis and protein extraction.
Hi Kate,
In our hands phosphatase inhibitors from Roche and GBioscience work well. But you can prepare by yourself 10mM stock solutions (1:100 working) of Na orthovanadate, Na molibdate, Al fluoride. Also you can go for microcystines, ocadaic acid which are very effective toward PP1/PP2 phosphatases (not sure equivalent in plants). If you still will have unreliable results I recommend TCA/aceton (15% v/v) fixation of your samples first at temp.
Thanks for all the responses guys! I'm going to try protein extraction using NaF in my extraction buffer, and I've ordered a test sample of Phos-stop from Roche, though due to my location (New Zealand) and Christmas madness it'll take a few weeks to get here. I've attached the Ponceau stain of the membrane shown earlier, and as you can see the total protein concentration is equal(ish) across lanes and the protein is not degraded. I'm also going to take multiple samples from the same plant to test if the variation is between plants, and I'm going to run the same aliquot of isolated protein out in multiple lanes to see if there's an issue in the gel-blot process. Another suggestion from a colleague is that a spray is a variable way of delivering glyphosate, so I'm going to try infiltration with as well. Individual responses below, and Christian Konrad I'd love to discuss this further! Please read the reply below.
Attila: eIF2A is found in plants, animals and yeasts under the name eIF2A, a quick google reveals that it's not the same as p53. eIf2A also doesn't bind to DNA, so I don't think that's the issue. I'd like to check total eIF2A content, but I've not yet been able to find a plant eIF2A antibody and the antibody made to animal eIF2A doesn't work with plant eIF2A. Sadly I have neither the time nor the funding to get an antibody made.
Jacqueline: I do all of my work on ice and spin the samples in a 4'C centrifuge. Total protein is consistent across lanes as tested via Ponceau stain. I do not have a non-specific plant eIF2A antibody. Phos-stop is looking like the way to go!
Ryan: Total protein looks good on Ponceau stain with clear bands and no smearing, so I suspect the issue is a low ratio eIF2A-P to total protein.
Christian: It's great to get a response from someone who's worked with eIF2A! First of all, what antibody did you use for total eIF2A? I used one raised against human eIF2A, but neither I nor others in my lab working with it could get it to work reliably - results were variable both between and within samples where the eIF2A-P antibody worked well. How did you quantify your eIF2A? What was your goal? Were you successful in answering your research question? I'd welcome any messages on this subject. Regarding boiling in SDS buffer, I've also found that necessary - previous work showed that without boiling the samples in SDS buffer I too got inconstant results. It's nice to know that that was due to dephosphorylation. What temperature and for how long did you boil your samples?
Wolfgang: As the attached Ponceau stain and unattached Qubit protein concentration measurements show, I have no issues with extracting the protein, and the protein is not degraded.
Mykhaylo: I'll definitely be trying Roche phosphoinhibitors, but they'll take a while to get here. In the mean time I'll be trying NaF, and if that doesn't work, I'll try some of the other inhibitors you've mentioned. I previously found that 30% v/v TCA/acetone fixation causes a decrease in the sensitivity of the antibody to eIF2A-P, although I am not sure why this is as the protein is denatured as part of the process normally. If necessary I will try 15% v/v. Thank you for your help!
Hi Saifudeen, my membranes are placed on a rocker during antibody incubation.
Hi Kate, I am glad I could help.
We used our own custom antibodies against both Toxoplasma eIF2alpha (TgIF2alpha) and the phosphorylated form. You can check the sequence in the our publication (Sullivan 2004). We saw no cross reactivity with human eIF2alpha so I am not sure if those antibodies would work for Arabidopsis.
When you have questions about human eIF2alpha I suggest you contact Ronald Wek at IU Indianapolis. He is an expert on this and we cooperated since he is down the hall from our department.
I normalized my sampled by loading equal amounts of total protein and then run my gel and blots to probe for TgIF2alpha (total and P form). I used the RIPA buffer and sonic action to prepare my samples. You will find the composition in Konrad 2011.
I used the NuPAGE system from invitrogen und their loading SDS-buffer supplemented with B-MeOH. I boiled my samples at 95C for 5 min in a heating block.
The goal of my work was to analyze what stress kinases are involved the response of Toxoplasma to stresses like nutrient deprivation. Toxoplasma expresses two GCN2-like kinases (TgIF2K-C and -D), which I studied. In addition, I tested inhibitors of eIF2alpha dephosphorylation and their effect of the parasite's proliferation and stage conversion. Toxoplasma con convert in a dormant stage, which is induced by cellular stresses and phosphorylation of TgIF2alpha appears to be involved in this step.
If you need further help, please feel free to ask me any question. You can also contact William Sullivan and Ronald Wek. We shared antibodies and protocols to start collaborations.
Best
Christian
Solved! Please see the question for the answer - it seems that my western was working perfectly, and the issue was that the plants themselves had different levels of phosphorylated eIF2 present in different leaves.
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