Hi Mita!

Does your protein sample contain EDTA (do you see a really strong smiling of your band along with the faster run)? If it does, add equivalent amount of Zn(NO3)2 (or MnCl2 if you use the old buffer system) before loading the sample to remove free EDTA that would chelate the Zn (or Mn) from the gel matrix. Also, do not use phosphate-containing buffers for sample preparation.

In case there is no free EDTA or phosphate in your sample: check the size of your protein on normal gel. Maybe there is some degradation or unusual form of your protein.

In case the problem is not the ones above: it is well known that all proteins run SLOWER on Phos-tag gel, not only the phosphorylated ones. If your protein runs FASTER than expected it is probably because it is a glycoprotein (full of negatively charged glycosyl groups). Highly phosphorylated proteins may run faster on a normal gel but certainly not on a Phos-tag gel.

Regarding the size markers: most of the protein markers will be distorted because they contain EDTA or are buffered with Tris-phosphate or some of the standard proteins are phosphorylated. Either use EDTA-free standard (difficult to find) or add equivalent amount of zinc (or manganese) to your favorite standard before loading (I have really good experiences with Bio-Rad’s Precision Plus Protein Dual Color Standard #161-0374 but add equivalent amount of Zn(NO3)2 (or MnCl2) before use because it contains EDTA). Adding the same amount of salts and other components your samples contain will eliminate any residual distortion. I hope this will help.

Good luck!

Peter

p.s.: I attached my Zn-Phos-tag protocol that works for me. I included a Troubleshooting section at the end of the document. Please read the recommendations for sample preparation as well which is a key to achieve nice separation.

https://docs.google.com/document/d/1LOi2hlF5D85DDC_MHocM1UjpW1yFc6GVH7fBMx1A8Jg/edit

Hi Mita!

Does your protein sample contain EDTA (do you see a really strong smiling of your band along with the faster run)? If it does, add equivalent amount of Zn(NO3)2 (or MnCl2 if you use the old buffer system) before loading the sample to remove free EDTA that would chelate the Zn (or Mn) from the gel matrix. Also, do not use phosphate-containing buffers for sample preparation.

In case there is no free EDTA or phosphate in your sample: check the size of your protein on normal gel. Maybe there is some degradation or unusual form of your protein.

In case the problem is not the ones above: it is well known that all proteins run SLOWER on Phos-tag gel, not only the phosphorylated ones. If your protein runs FASTER than expected it is probably because it is a glycoprotein (full of negatively charged glycosyl groups). Highly phosphorylated proteins may run faster on a normal gel but certainly not on a Phos-tag gel.

Regarding the size markers: most of the protein markers will be distorted because they contain EDTA or are buffered with Tris-phosphate or some of the standard proteins are phosphorylated. Either use EDTA-free standard (difficult to find) or add equivalent amount of zinc (or manganese) to your favorite standard before loading (I have really good experiences with Bio-Rad’s Precision Plus Protein Dual Color Standard #161-0374 but add equivalent amount of Zn(NO3)2 (or MnCl2) before use because it contains EDTA). Adding the same amount of salts and other components your samples contain will eliminate any residual distortion. I hope this will help.

Good luck!

Peter

p.s.: I attached my Zn-Phos-tag protocol that works for me. I included a Troubleshooting section at the end of the document. Please read the recommendations for sample preparation as well which is a key to achieve nice separation.

https://docs.google.com/document/d/1LOi2hlF5D85DDC_MHocM1UjpW1yFc6GVH7fBMx1A8Jg/edit

Hallo everyone, I will bid on Mita's question as I am routinely using Phos-Tag since 2007. Peter: I am not using the Zn improved version, but rather the original protocol with Mn. I followed your text and I saw that I am doing practically the same with the exception that I am using classical Tris-glycine elpho system. However my results are rather ambiguous, although I always run positive and negative markers on the same gel (that is known phosphorylated protein before and after lambdaPPase treatment). Therefor I trust that I may have an uncontrolled transfer efficiency problem. Just today, I tried to wash my PhosTag gels with phosphate buffer (25 mM, pH 8) and transfer was a lot better. Can you please send me your feedback on this??? Please note that I am working with crude extracts. Could you please tell me if it is essential to use the MOPS system you describe???

Hi George!

What do you mean by ambigous results? Is it only the transfer you have a problem with?

Earlier I used the Mn-based system but then I tried the Zn-based and I stayed with it (sharper bands, inclusion of an antioxidant in the buffer which is incompatible with the standard buffer, etc). I also experienced this inconsistent transfer problem (especially with the standard glycin-based transfer buffer). Therefore I also tried to wash my gel with a buffer containing phosphate before transfer (to "elute" the proteins) but I didn't see any improvement to the EDTA-only version. The transfer buffer I use now (NuPAGE transfer buffer) already contains 1 mM EDTA, so the EDTA is in the buffer during the transfer (unlike in the standard protocol when you wash the gel once with EDTA and then without it). I think it is a good thing to have EDTA in the buffer during the transfer and I do not see why you cannot include EDTA in the standard glycin-based transfer buffer as well (it will change the conductivity of the buffer somewhat, though). The transfer time also matters, at least in the case of large proteins (I had a 125 kD protein to detect...). So I did the transfers overnight in the cold room and it was fine. If you do not have a large protein then 1-3 hrs should be enough.

Regarding the MOPS buffer, I do not think it is essential. The standard buffer system and MnCl2 should be all right but according to the authors the Zn-based methods is more reliable (the Phos-tag based phopshopeptide enrichment kit also uses Zn). Also as I mentioned, you cannot put antioxidant into the standard buffer to prevent reoxidation of the SH-groups because at that pH it doesn't work and my protein contained disulphide bridges, so I stayed with the Zn method. My colleague, who didn't use Phos-tag gels, only standard ones immediately switched to this NuPAGE buffer system after I showed it to her because she could make extremely nice westerns with it. Well, certainly it is a bit more expensive than the standard system but when you see the nice results...

I hope it was helpful. If not, just ask!

Cheers!

Peter

Dear Peter,

I thank you with all my heart. Ambiguous means that I get my protein of interest phosphorylated sometimes and sometimes not (sometimes the same sample). I am dying to use your entire protocol as this will save me from a lot of trouble. Just one question though: Is EDTA safe for electrophoretic transfer? Because from what I figure out Koike et al, dont like EDTA during transfer.

Hallo Peter once again. I read the entire protocol you have posted and I have some minor questions on it:

1. Is it absolutely necessary to use zinc nitrate, or you can use zinc sulfate or zinc chloride as well?

2. I saw you highlighted the omission of SDS from both stacking and separating gel. Does this mean that ommiting SDS is absolutely essential?

Hi George!

1. Zinc NITRATE is essential, according to the authors of the original paper.

2. In the NuPAGE buffer system (where the same buffer is used for the stacking and the running gel) there is no SDS in the gel, only in the running buffer. They say it is unnecessary in the gel and it also results in sharper bands. As I remember I have read somewhere that SDS can be omitted from the standard Tris-glycin gel buffer as well but I have never tried that.

3. Regarding EDTA in the transfer buffer: as I said earlier adding EDTA will change conductivity (the buffer will be heated more). However, in the NuPAGE transfer buffer there is 1 mM EDTA by default, so at least in that buffer it is not a problem. I guess 1 mM EDTA is not too much, you can try to add it to your buffer. Oh, and I put only 10% methanol not 20% as in the standard protocol. Methanol shrinks the gel inhibiting the transfer of large proteins.

4. I had problem with that phosphatase treatment as well (the same ambigous results) in the beginning but later it worked fine. I used NEB lambda phosphatase (I tried others but it is the best). I always put the enzyme and the MnCl2 directly to the extract (I do not add extra lambda phosphatase buffer) therefore I use an extraction buffer which is close to the lambda phosphatase buffer (i.e. Miltenyi Lysis buffer or similar). Obviously you have to omit EDTA and phosphatase inhibitors from the extraction buffer. However, if you want to include EDTA in the extraction buffer then you have to add more MnCl2 into the phosphatase reaction to remove free EDTA (if you want to include an 50 mM EDTA control which blocks phosphatase activity then you have to add MnCl2 to this sample only before loading). I do not know what loading buffer you use but commercial ones usually contain EDTA. Check the exact amount it contains and make sure that before loading you add the necessary amount of MnCl2 to bind free EDTA. Always check the temperature of your heating block or thermostate (do not believe the digital screen!). When I had really bad results I figured out that in the digital heating block the temperature was 5 degree less (25) then the shown value (30)... Oh and one more thing: I realised that is it essential to prepare an "mock" enzyme storage buffer (which contains EGTA and MnCl2) with the same composition as in the real enzyme solution. Then you have to add the same amount of mock storage buffer to the "untreated" sample which you run next to the "treated" one. Otherwise the neigbouring bands will be distorted like hell...

Thank you Peter for your valuable inputs. I actually add Phos-tag to the gels while I make them. And I use the Mn system. So far, its pretty good. I am following the protocol found in this link http://www.wako-chem.co.jp/english/labchem/product/life/phos-tag_aal_guidebook/pdf/gb.pdf

Prior to running western blot, I soak the gel in a transfer buffer with 1 mmol/L EDTA for 10min with gentle agitation and next in the transfer buffer without EDTA. I could see my protein of interest phosphorylated and unphosphorylated with a shift in molecular weights. I would still like to improve upon the bands that I see. So, I will definitely try adding Mn to the sample before loading. Thank you.

Thank you Peter for your protocol! I'm just starting to work with Phos-Tag gels and was thinking of improving the technique with Zinc. I could only find Zinc Nitrate Hexahydrate in our lab though. Can this be used as well? Thanks!

Hi Sarah!

I think the hexahydrate salt is just as good as the others. Only the final concentration of zinc that matters.

Good luck!

Thank you Peter! I am doing the gel tomorrow, so I'll let you know how it goes! Thank you for your help!!! :)

I appreciate any feedback, especially the positive ones... :D

Hi, Peter. This is Eiji Kinoshita in Japan.

Thank you for your nice protocol. It is really wonderful.

In another thread, I would like to introduce your protocol to Phos-tag users.

I want to add only one thing regarding the counter anion of zinc.

You can use zinc chloride in the new version.

However, for using manganese ion in the older protocol, chloride is essential.

Because manganese nitrate is precipitated as Mn(OH)2 in an alkaline Laemmli' s buffer solution, the gel becomes muddy and Phos-tag loses the function.

The same phenomenon is also observed in the case using manganese acetate.

I believe you will never see such phenomenon in the new version of neutral Zn(II)-Phos-tag gel.

Anyway, the opinions from the Phos-tag users become my power.

Many thanks!

Dear Mr Kinoshita,

Thank you very much for your correction! It is a valuable information since zinc chloride is probably cheaper and more widespread.

Yes, I think so!!

Hello,

I would like to show you guys my best results. Unfortunately I could not get it better and my problem is that the bands are not sharp, so even if IRE1 has only one phosphorylation site it produces multiple bands.

Any hints, please?

5% Phos-tag gel (minigel), 25uM of substrate and I run the gel for 3 hours at 100V in TGS buffer.

Thanks

Hi,

I guess here is the expert guys about Phos-tag gels!

I am going to start using these gels

I wonder if the lysis buffer I will use would affect the migration of proteins on SDS-PAGE

am gonna use

50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1.5 mM sodium orthovanadate, 50 mM NaF, 10 mM pyrophosphate, 10 mM glycerophosphate and 1% Triton X-100,complete protease inhibitors (Roche) with EDTA).

appreciates your feedback

thanks

Hi, Mahmoud,

I have no experience using the lysis buffer, but pyrophosphate is strongly captured by Phos-tag. So, it may be a major competitor for your target in the Phos-tag gel.

Why do you need 10 mM pyrophosphate and 10 mM glycerophosphate for your sample? I think that 1.5 mM sodium orthovanadate and 50 mM NaF are too enough as phosphatase inhibitors.

Thanks.

Thanks Eiji for your reply,

yes i guess i will omit both

appreciates your help

Mahmoud

Does anyone know how much Zn(NO3)2 or MnCl2 to add to the standard marker, so that it runs correctly? thanks!

MnCl2 should quench exactly the amount of free EDTA in the standard marker

Which marker do you use?

Biorad pre-stained sds-page low range marker#161-0305

So if i load 5ul of standard, i should add 5ul of 10mM MgCl2 to it?

Hi Sarah!

I am sorry for the late answer but for some reason I didn't notice you added a response.

To answer your question, I think you should add 1 uL MnCl2 to 5 uL of your marker (according to the product sheet of your marker it contains 2 mM EDTA).

Hey Peter,

Thanks for your response. I wasn't sure what to do, so I I actually did add 1ul MnCl2 to the 5ul marker.... It turned out for me that it still didn't transfer to the PVDF membrane. Maybe it's because of the membrane that it won't transfer...or the 10% methanol transfer buffer...or maybe the concentration of the gel (I'm using a 4% stacking and 10% resolving).

Any suggestions I would appreciate! Thanks :)

Hi, Sarah

Regarding the efficiency of transfer, my opinion is described below.

I think it is effective to add SDS to the transfer buffer you usually use. Please determine an optimal concentration of SDS of 0.05–0.2% (w/v) for your target. However, the efficiency is dependent on the nature of proteins.

So, please notice that some hydrophilic (or small) proteins may pass through the blotting membrane.

We usually use 0.1% of SDS in our experiments.

This transfer problem was observed by many people. What is the size of your protein you want to resolve? I used 7% (37.5:1) acrylamide gels for 150 and 65 kDa proteins with good results. The 37.5:1 acrylamide:bis-acrylamide ratio means larger pores in the gel at the same percentage. If you use 29:1 stock it is worth trying 37.5:1. Also I used the NuPAGE buffer system (bis-Tris gel buffer, Tris-MOPS running buffer). The Invitrogen's transfer buffer contains 1 mM EDTA (during the gel washes you can use more than 1 mM EDTA) – so there is EDTA during transfer – and only 10% methanol which results in swelling the gel and therefore bigger pores (and reducing agent that prevents artefactual SH-bonded aggregate formation during transfer). It doesn't contain SDS. According to the literature adding SDS to transfer buffer could help transfer of large proteins (which tend to lose SDS because of washes with methanol). However, I didn't observe any improvement when I added SDS (actually SDS interfers with binding of proteins to the hydrophobic surface of PVDF). Reducing the amount of methanol from 20% to 10% was better for me. I also performed overnight transfer (wet blot, not semi dry) in a cold room with 150 mA constant current (Bio-Rad Mini Protean System).

Finally, maybe different markers transfer differently. The Bio-Rad marker I used (Precision Plus Dual Color) transfer was quite good. Did you stain the gel after transfer to check if proteins remained in the gel (not just the marker)? Did you stain the membrane for total protein after blotting?

Oh thank you, thank you, thank you!!!! I will apply these suggestions immediately to our protocol! Yes I am staining the gel and there are still proteins on the gel. I did not stain the membrane tho.... And currently we are adding SDS to the transfer buffer.

Dear All,

I have tried both Mn and Zn based phos-tag separation. Mn gives a nice separation. However, the Zn based phos-tag has some funny behavior during running in the gel. After blotting and Ponceau staining, Most of the protein is stucked at the most upper region of running gel.

The gel concentration I used is 8%, and Phos-tag is 25 uM.

Could anyone help me to figure out the reason for Zn based phos-tag gel? Thanks!

Here is the Ponceau staining picture

Dear Peter and Eliji,

       Thanks for the great protocols you both have posted. Using them (mostly Peter's since it is written in an easier-to-follow language but Eliji's transfer buffer), I was able to get great bands from my first Phos-Tag gel! 

         I just had one quick question. I see that Peter's protocol is optimized for larger proteins. I would like to check phosphorylation of a smaller protein, survivin, ~17 kDa, and I was wondering what tweaks you would make. I was thinking in terms of the acrylamide ratio (29:1 instead, perhaps?) and percentage, as well as the transfer conditions (two hours at 100 V instead of overnight at 150 mA?). Any tips you can give would be greatly appreciated! Thanks and take care!

Hi, Joshua.

I think you should optimize the concentration of acrylamide.

I recommend using ~12.5% (w/v) polyacrylamide gel for low-molecular-mass proteins of ~17 kDa.

As another strategy, I have used a precast Phos-tag gel, SuperSep Phos-tag, which is commercially available from Wako, for analyzing a smaller protein, histone H3, ~15 kDa. The precast gel is a 12.5% (w/v) polyacrylamide gel containing immobilized Zn(II)–Phos-tag (50 µM). Although no information on the neutral-pH buffer system used in the precast gel is provided, the manufacturer recommends the use of a general Laemmli’s electrophoretic running buffer consisting of 25 mM Tris, 192 mM glycine, and 0.10% (w/v) SDS (Tris–glycine buffer). I examined the potential usage of a Tris–Tricine buffer (50 mM Tris, 50 mM Tricine, and 0.10% (w/v) SDS) as an alternative running buffer for the SuperSep Phos-tag precast gel system in the analysis of low-molecular-mass phosphoproteins. Compared with Tris–glycine, the Tris–Tricine running buffer improved the resolution of 8.8–35 kDa phosphoproteins and phosphopeptides. Thus, use of this good tool is worthy of consideration for a laborsaving, timesaving, and substantial screening in the reliable detection of low-molecular-mass phosphoproteins. I attached our data file for the analysis of histone H3 and our paper on SuperSep Phos-tag.

Good luck!

I think the Phos-tag SDS-PAGE protocol using Mn is very simple because it is compatible with the buffer reagents used for conventional Laemmli’s SDS-PAGE. So, I recommend using Mn as the first experiment. When you want to analyze the phosphorylation status of your interested target in more details, you should select the protocol using Zn. The Zn protocol is improved in most cases. Good luck!

If the phosphorylation is minor. Like 4 sites. Could the differences be seen using this technique? thanks

Yes, I want to believe in such a case. Please try. Many thanks.

Hi Peter ,

I followed your protocol to run the phos-tag gel, except  used ZnCl2 instead of Zn (NO3)2 as it was mentioned in the thread we could use ZnCl2 as well.

However, I observed on both coomassie staining the gel and ponceau staining my blot that the proteins were mostly stuck at the top of the separating gel. Also, even though I added equivalent amount of ZnCl2(2.5mM) to my Novex protein marker, it didn't run well and seemed to be stuck at the top of the gel too or didn't run as expected. Any help is appreciated.

Thanks

Swati

I am sorry to interfere from the side. This is Eiji.

It is recommended that tests should be conducted by using microM level of concentration of Phos-tag (e. g., 5–25 microM) for complex samples that contain various phosphorylated and nonphosphorylated proteins, such as cell lysates.

How about your gel?

Thank you for  your response. The only reason I directed to Peter was because I followed his protocol, so  he may have encountered it and be able to help me troubleshoot.

I use sarcoplasmic reticulum membrane fractions and will try using lesser concentrations of Phos tag. I will try the different concentrations and let you know how it goes.

Kayleigh,

       The protein marker bands do not run as they normally do in a SDS-PAGE, due to the different gel chemistry, so it's not reliable to estimate protein sizes based on those bands. For that reason, after transfer, I blot the whole membrane, not just the predicted area. 

       Keep in mind that the band given by the phospho-specific antibody will represent a protein form that has at least one phosphorylated site, so its movement through the gel will be retarded compared to non-phosphorylated protein.

       A much better control, which I have done and worked well for me (except in the case of MYC; it starts getting ubiquitinated right away!), is to treat a fresh lysate, with no phosphatase inhibitors added, with phosphatase, and run that sample right next to your sample(s) of interest. That will show you the where the non-phosphorylated band lies. 

      Let me know if that works out for you!

Hi, Kayleigh, thank you for using Phos-tag. 

By some possibility, your case may be caused by inefficiency of transfer of the Phos-tag gel. We have found that the sensitivity of Western blotting after Phos-tag SDS-PAGE is unfortunately inferior to that after conventional (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane.

We previously described tips on improving the efficiency of electrotransfer of target phosphoproteins from Phos-tag gel. Please see the paper (http://www.ncbi.nlm.nih.gov/pubmed/25266391).

We have an experience of the analysis for STAT1 by using Phos-tag SDS-PAGE. The data is attached. This protein is constitutively phosphorylated under our conditions. After some stimulation, it is further hyper-phosphorylated.

The protocol is briefly described below.

SDS-PAGE: Zn(II)-Phos-tag SDS-AGE under neutral pH conditions.

Phos-tag: 25 µM

Gel: 6.5% w/v T

Blotting: wet-tank method

Transfer buffer: 25 mM Tris containing 192 mM glycine, 5% w/v MeOH, and 0.1% w/v SDS.

Pretreatment of Phos-tag gel before blotting: No need!

Membrane: PVDF

Antibody: CST #9172

Good luck!

Eiji

Hi, Kayleigh, I think the Mn(II) procedure is also good as the first your choice.

In genral, the separation in the Zn(II) procedure is better.

Regarding STAT1, we do not have an experience by using the Mn(II) procedure.

If you have any troubles, please contact me.

Good luck!

Eiji

Hi all,

I'd like to throw in a related question. Which phosphatase inhibitors in cell lysis buffer are in your experience compatible with Phos-Tag PAGE? The method guidebook (http://www.wako-chem.co.jp/english/labchem/journals/phos-tag_GB2013/pdf/Phos-tag.pdf) says that vanadic acid can distort the bands and recommends omitting it from samples (which applies to sodium orthovanadate as well, I guess). On the other hand, pyrophosphate and glycerophosphate are listed as incompatible with Phos-tag agarose but not specifically mentioned to interfere with PAGE.

Regards,

Jaakko 

Hi, Jaakko

I usually use a RIPA buffer, for example, consisting of 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 0.25% (w/v) sodium deoxycholate, 1.0% (v/v) Nonidet P-40, and 1.0 mM EDTA. And in the buffer, 1.0 mM phenylmethanesulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 µg/mL leupeptin, 1.0 mM Na3VO4, and 1.0 mM NaF are further added before use as a lysis buffer. Commercially available Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail can be also used for Phos-tag SDS-PAGE. It’s no problem at all as far as the use of common-sense concentrations of them. An important thing is that their compositions are the same in all the samples for the analysis of Phos-tag SDS-PAGE.

In the Phos-tag chromatography method, inorganic phosphate or pyrophosphate is used as an elution buffer. I think that the manufacturer will recommend so that to avoid a leak of phosphorylated targets.

Good luck!

Eiji

Dear Eiji Kinoshita,

would you mind to clarify the difference between old Vs new protocol, please?

Thanks a lot!

Dear Davide,

Thank you for your message. The differences between the two protocols are metal ions for complexing Phos-tag ligand and the buffer systems that are used for Phos-tag SDS-PAGE.

For the old protocol, a polyacrylamide-bound Mn(II)–Phos-tag and a conventional Laemmli’s buffer system under alkaline pH conditions are used.

Please see our previous papers for the procedure of Mn(II)–Phos-tag SDS-PAGE as follows:

Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol. Cell. Proteomics 5, 749–757 (2006).

Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis. Mol. Cell. Proteomics 6, 356–366 (2007).

On the other hand, our new protocol uses a dizinc(II) complex of Phos-tag acrylamide in conjunction with a Bis-Tris-buffered neutral-pH gel system. Please be referred to our previous papers for the procedure of Zn(II)–Phos-tag SDS-PAGE as follows:

Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics 11, 319–323 (2011).

Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions. Proteomics 12, 192–202 (2012).

I attached our recent protocol article, and I hope that it will be helpful for your works.

Good luck!

Eiji

Thanks a lot for your reply, Eiji.

Would you mind to help me with the following questions, please?

 1) Polymerization: should I let the separating gel (Zn-Bis-Tris protocol) for 16h (overnight) or 20 min are sufficient?

2) Markers: It is my understanding that pre-stained markers (from BioRad, for instance) are to be avoided.

3) your RIPA buffer contains 1mM EDTA. Do you recommend to add some Zn to 'neutralize' the chelating agent? Or is it better to use a simple Laemli sample buffer right away?

Thank you again!

D

Hi, Davide,

Thank you for your message! it is the same as your above description, so I replied here.

1) If the polymerization of the gel is complete for 20 min,  I think that's OK. When polymerize it overnight, please protect it from drying.

2) Yes, basically I agree with you, but recently the manufacturer recommends the use of certain pre-stained markers (Wide-View Prestained Protein Size Marker Ⅲ, Wako, 230-02461) for Phos-tag SDS-PAGE. The markers show a good banding image without waving or tailing of protein bands. I attached the data. If you need pre-stained markers for your analysis, use of this might be worthy of consideration for you.

3) No, you don't need to add Zn. There is no problem for using RIPA buffer containing 1mM EDTA for Phos-tag SDS-PAGE, because the lysate sample for the analysis actually has an only small amount of EDTA. As you suggested, I think that the use of Laemmli sample buffer as a lysis buffer is the most simple.

Good luck!

Eiji

Thank you again, Eiji.

I am actually using your protocol at this right moment. I have got a doubt about the possible interference of genomic DNA with the run.

Would you mind to send me your protocol for benzoase treatment? Can I use the nuclease directly in my Laemli buffer (your recipe)?

Thank you SO much.

D

Hi, Davide.

Yes! As you mentioned, genomic DNA in the lysate sample sometimes interferes in electrophoresis. We usually use benzoase, which is directly added in a sample-loading buffer, for the analysis of Phos-tag SDS-PAGE.

Best wishes!

Eiji

Hi all, I'm having a lot of trouble seeing any signal from my antibody on my blot! I am probing for total S6 on whole cell, ESC lysate. I have optimized transfer buffer and added 0.1% SDS. There is no protein leftover on the gel. I see okay bands on my 0um gel but on the 30um phostag the signal is very low. I was wondering if you had any tips on improving this. I can see faint bands so I know there's separation. However, I would like to make the bands darker like your STAT5 blot!  Thanks in advance. 

Dear Mansi,

I understand that you have checked no protein on the Phos-tag gel after electrotransfer.

If so, you next should try to decrease the concentration of SDS, which is added to the transfer buffer.

Good luck!

Eiji

Not to disagree over much with the developer of Phos-Tag, but I have found that for proteins less than 100 kDa, there is no need for SDS in the transfer buffer for Phos-Tag westerns.

A bigger factor might be the duration of your transfer. I transfer overnight for a Phos-Tag western at 30 milliAmps and that works great, for proteins in the 40-130 kDa range.

I attached our data. For details, please see PMID:25266391.

Many thanks!

Eiji 

That is a good systematic way to analyze the different parameters. It does seem that, for Zn Phos-Tag gels at least, perhaps SDS in the transfer buffer helped transfer some species and not others of the target protein? 

First of all, thank you so much for your fantastic advice.

I am currently using phos-tag gels with Mn and I use the old school Tris-Glycin (10% Methanol) transfer buffer for blotting on the nitrocellulose membrane. Fortunately, I had really good results for proteins around 60 KDs, but I can not see any transferred proteins on the membrane which is stained with CBB R250 under 35 KDs (they do not present in the membrane too). I also tried the same transfer buffer including 0.1 % SDS and it only causes that big proteins go through the membrane, but nothing improved on the membrane for small proteins.

Since I am not able to use precast phos-tag gels as Eiji mentioned, do you have any other suggestion to improve this blotting before switching to Tris-tricin buffer?

Thank you