hi

try to use stable cell transfectant or equal rate of transient transfectant cells ( such as 40% transfection rate for 3 cell lines SH,HEk,PC12). you can check 20ug loading of cell lysate .

Article Improved Immunodetection of Endogenous α-Synuclein

good luck

Hi Reza, Thanks!

In fact, I use equal transfection rate cells in my experiments. And about the paper, I already read it and because that I started to fix the membrane with 0.4% PFA before blocking and everything.

Any other suggestion? :)

Hi Ana, I'm having some difficulty following your experiment, a bit too many uncertain points... then of course giving you any suggestion is difficult. So let me do some troubleshooting and ask you a few questions: 1) How is your plasmid engineered? Who is controlled by the IRES, the GFP (I hope so) or the alpha-synuclein? (This could explain why you lose expression of the GFP in the full vector, if the Cap-dependent translation is working well, the IRES-dependent translation will be less efficient. But if there is no CDS before the IRES, there is no competition on the mRNA and the IRES-dependent translation will work better. IF is more sensitive than WB, for this reason you probably detect some fluorescence at the microscope. Moreover a smaller plasmid might also be better transfected into cells, so more copies could enter a cell or more cells be transfected. Lastly, they are two different plasmid preps and one might have come out cleaner, one less, affecting differently the transfection efficiency - seems the case of the GFPblot image, top blot, see point 2) 2) Which cells are expressing endogenous alpha-synuclein? I can see that SH cells do (you see the doublet at 20 kDa, the top band being your His-tagged version, in the Nooligomers picture, top blot). This means that in the top blot of the GFPblot image you did not transfect the cells, or the transfection efficiency was too low (few cells, or too little plasmid per cell), and the His-tagged band is missing from the WB. Anyway the GFP band in the C- is already pretty weak...). PC12 cells also express alpha-synuclein, but it's rare to see your His-tagged version and the endogenous in these cells, anyway in the bottom gel of the GFPblot image something went wrong with the WB or the PC12 extracts, all signals are too low, and when a-synuclein monomers are so weak you can't expect to detect GFP expressed from an IRES. Finally HEK cells... impressive, they seem to express low levels of a-synuclein, all your C- controls show a weak band at the right height. Anyway, it's a good control to verify that you are transfecting your cells. Secondly, they never show tetramers... Possibly the formation of the multimers is concentration-dependent... 3) The multimers: What you see at 75 kDa, ONLY in PC12, I personally doubt it's tetramers, it seems a non-specific band from the Ab recognizing something in PC12 (in the Notubulinin PC12 image you have this band in the C- where you don't even detect the tranfected monomer nor the endogenous...). Anyway, if you have sure evidence that it's tetramers, keep in mind you see them only in PC12. You are boiling the protein extract in SDS and you are running a denaturing gel... it's difficult to imagine aggregation of such a small disordered peptide... Also what you call dimers, which you detect at the same level in transfected and C- samples or with stochastic intensity levels.... very suspicious... Have you ever checked your Ab on a a-Syn KO or KD extract? I would do it... or test a different Ab... So, you are right, it looks quite chaotic. You also seem to have protein degradation, at least sometimes (the 25 kDa tubulin band in the bottom blot in the Nooligomers figure). To start, you can try to improve a few things in the protocol: 1) My suggestion is to lyse the cells directly on the plate on ice: remove medium, wash twice with warm PBS like if you should passage the cells, remove thoroughly the PBS and place the plate on ice. Then add the 100 ul of ice-cold lysis buffer on the cells in the well. Pipette to help the lysis a few times in the well and transfer in tube. Do not vortex, there is no need, the RIPA is strong enough (in fact the TritonX would also suffice) to lyse. You can shake the tube once in a while with your fingers or pipette while you incubate on ice. Incubate 15 min on ice. Never bring native protein extracts at room temp, always on ice. 2) Proteins are delicate! Thawing and freezing degrades them quickly, denatures them and induces aggregation depending on the protein type. My suggestion would be if you only need them for WB, immediately after lysis quantify while keeping the lysate on ice and then denature them with Laemmli and boil. Denatured proteins dstill degrade when thawing and freezing, but more uniformly and less. Store in 50 ug aliquots at -20 (so that you thaw and freeze the only 5 times). Anyway, if you wish to freeze them native, then you should shock-freeze them in liquid nitrogen. 3) WB. I had quite bad experience with semi-dry blotting. It is very uneven... and I believe you are experiencing this quite clearly. My suggestion would be to try a few blots with the submerged method. It's longer (70 min at 110 V), it uses more buffer... But you will be surprised by the result... Then don't forget what I mentioned above... test a different Ab or try to obtain or produce a-Syn knock-out or knock-down protein extracts... Good luck.

Hi Pietro!

First of all, thank you very much for taking your time to answer me. I really appreciate it.

I'm going to answer you by points as you did :)

1) Yes, GFP is controlled by IRES and the GFP plasmid (without any alpha-synuclein) doesn't have anything before, so no competition. I'm going to do qPCR to check if the level of expression of alpha-synuclein and GFP are similar in cells transfected with different plasmids. Therefore, I will be able to say if the translation and folding of the protein are being different because the mutation I'm introducing.

2) If I believe that the band at 75 kDa in PC12 is alpha-syn, I would say that HEK, SH, and PC12 are expressing endogenous alpha-synuclein. SH and HEK monomers and dimers, and PC12 dimers and tetramers (Although sometimes I didn't see dimers). By the way, when I incubate with anti-6xHis I only see monomers, so dimers or other multimeric forms are endogenous.

In GFP blot, the first lines are sorted cells and I only see exogenous alpha-synuclein (his tag). You said that something went wrong because everything is weak, but the fact is that it was the 3rd time I develope the membrane and with anti-mouse to detect tubulin and GFP. That's the reason why alpha-synuclein is so weak. But yes, the reason for not seeing GFP in my transfected samples is probably due to what you said in point 1).

3) Multimers: You suggested that tetramers are concentration dependent. I've tried 2.5 ug, 5 and 10 ug of lysate (the maximum I'm using in my blots) and always was the same. The only thing that affected was if the fixation or not of the membrane, and the use of a stronger lysis buffer what destroyed the tetramers in PC12.

Also, I'm going to run also native gels to check if I'm able to see the multimeric forms.

I'm sorry because I wasn't clear with No-tubulin blot. There, the samples are treated with cycloheximide but they are not transfected at all. So, the bands we are seeing are always endogenous alpha-synuclein.

About degradation of the samples, I'm keeping them always on ice or -20ºC, but as you said I will start to store them already with Laemmli. The point is that this blots that I showed here were with fresh samples, not reused before. Anyway, I will start to lysis the samples directly on the plate on ice.

Thank you very much again!!

Best,

Ana.