Some cell lines give 2 bands for ERK1/2 and others just show one merged band, despite being processed alongside one another using the same protocol at the same time. Perhaps double check literature on the previous results your cell line.

I have used the cell signalling technology antibodies for phospho and total ERK (both rabbit), they give fantastic results. The ERK1/2 signal can be quite strong and easily saturated - I haved the protein loading compared to samples that I wanted to probe for Akt. Also, 3% BSA in TBST was sufficient for blocking and antibody incubations, make sure your primary antibodies are left on overnight at 4 degrees with agitation, and better resolution between the bands at 4-20% gel when compared to a straight 10% gel. The power you are running the gel at might influence the clarity of your bands - I recommend 150-170V for about 1hr. When running faster, the bands can become more blurry and there is lower resolution between such close-MW bands.

Regarding the methods for stripping membranes to reprobe for total ERK subsequent to phospho-ERK, I recommend the Mild Stripping protocol from abcam. Use 2 x 5-7min incubations with this buffer. See this link below for the recipe:

http://www.abcam.com/index.html?pageconfig=resource&rid=11353

There is minimal protein loss and the reprobes come out beautifully. I only recommend reprobing once, doing it multiple times is not a good idea in my experience.

The problem might be in gel running. Use 15% gel (if you use 12 or 10% gels may cause merging of the bands). Second, use different protein concentrations in 15% gel because the liver sample usually clogs in the wells (lower the protein concentration better the separation of ERK bands and lesser clogging in the wells... on contrary, more the protein concentration, both ERK bands might merge or may result in clogging in wells (only for liver samples). Finally, check in uniprot (old: Swissprot) protein database that whether the animal model you work on has two ERK isoforms or not.

Other procedures you mentioned above looks ok..!!

First, Perhaps is the gel concentration. Use 15% gel to separate the bands and second, use lower protein concentrations in gel because proteins are collapsed in the weels.

i think you should use the 12.5% gel and run it at 100 voltage for 1.5 hours. and for the 1ry Ab why do not you use it in (PBS tween 1%+ 0.01% of your blocking solution) and keep it O/N at 4 degrees

To all :-)

I used 12.5% gel and loaded 16 ug (4ug/ul) protein sample

And I think they are needed more running time.

(because of merge band you said)

Thank you every body X)

To Valentina Sala

I'm so sorry about I voted your reply "downvote" ;-(

I really have poor mouse control ;-(

It's not real.

Thank you for your answer :-)

If you used large quantities of lysats you will see only one band for phospho-Erk so I advice you to use 30 micro gram of protein and then follow your procedures, you will see the two bands of phospho-Erk

Also, because of the high amount of albumin within whole liver lysate, it become difficult to blot for proteins that fall within that range (40-50kD) by conventional SDS-PAGE; try blotting for actin in the liver and you'll see what I mean

Even when 10 or 15% gel should work you can try with gel gradient 4-20%. Second, make sure your antibody is against both isoforms and not only against ERK1 or ERK2. Good luck

My experience is that the apparent protein sizes on gel/blot for ERK1/2 vary between species, I have seen a lot more separation between 1 and 2 in rat tissue (blood vessels) as compared to HUVEC and mouse blood vessels. Likewise for the ratio ERK1/ERK2, which is tissue and species dependent. In mouse blood vessels ERK1 is dominant, whereas ERK2 is sometimes barely detectable. I use 8-12% gels and get good separation, but a lot less protein (1-2 micrograms, no kidding) and still get a good signal (with ECL advance). Have you determined the optimal amount of protein for (p)ERK1/2 in your assay?

I can tell you that for us with mouse cells the Cell Signaling p-ERK and ERK WB-ABs work excellent. We use premade (bought 4-12% gels). Dilution of the primary is 1:500-1:1000. I usually load between 20-40ug of protein depending how much i just have. the more, the easier the detection but 30ug should be totally fine. Which AB are you using?

Which antibody exactly are you using? Some antibodies recognize only Erk2.

An a normal 10% gel is good enough for P-Erk and Erk detection.

pERK and Total ERK bands cannot be resolved so easily by western. Instead I highly recommend serial probing.

Briefly, probe the western blot membrane with pERK antibody, go ahead and image the signal.

Then strip the membrane using a stripping buffer using the reagent below followed by washing several times with western transfer buffer, then blocking.

http://www.piercenet.com/browse.cfm?fldID=F80AEC89-FFFF-488B-917E-E7F50837E9E9

Now probe the western blot membrane with total ERK antibody and image it.

Now you will have phospho ERK image on one film and total ERK on another.

You can do densitometry using a scanner and determine the ratio of pERK to total ERK to show if there is a change in ERK activation among your treatments.

If you have more questions, please feel free to post on this thread again.

Good luck, RJ

Ah I missed out on basics of blocking. Use 5% BSA in TBS-T to block. There are phophatases in dry milk that might cleave the Phospho moiety during the blocking step, so please avoid using nonfat dry milk.

Also please try out different exposure times for signal capture on the film, your data is there ! it is just a matter of signal to noise ratio that bothers all of us.

Some cell lines give 2 bands for ERK1/2 and others just show one merged band, despite being processed alongside one another using the same protocol at the same time. Perhaps double check literature on the previous results your cell line.

I have used the cell signalling technology antibodies for phospho and total ERK (both rabbit), they give fantastic results. The ERK1/2 signal can be quite strong and easily saturated - I haved the protein loading compared to samples that I wanted to probe for Akt. Also, 3% BSA in TBST was sufficient for blocking and antibody incubations, make sure your primary antibodies are left on overnight at 4 degrees with agitation, and better resolution between the bands at 4-20% gel when compared to a straight 10% gel. The power you are running the gel at might influence the clarity of your bands - I recommend 150-170V for about 1hr. When running faster, the bands can become more blurry and there is lower resolution between such close-MW bands.

Regarding the methods for stripping membranes to reprobe for total ERK subsequent to phospho-ERK, I recommend the Mild Stripping protocol from abcam. Use 2 x 5-7min incubations with this buffer. See this link below for the recipe:

http://www.abcam.com/index.html?pageconfig=resource&rid=11353

There is minimal protein loss and the reprobes come out beautifully. I only recommend reprobing once, doing it multiple times is not a good idea in my experience.

I really failed with ready made stripping buffers also, I lost all of my protien.

I have a nice homemade stripping buffer: 1.5 g glycine, 0.1 g SDS, 1 ml TWEEN-20, up to 100 ml with distilled water, set pH to 2.2. Wash and agitate the membrane with: 10 ml PBS 10 min, 10-20 ml stripping buffer 10 min (vigorous shaking), 2 times 10 ml PBS 10 min, 2 x 10 ml blocking buffer 10 min. Continue WB from blocking. Use it freshly.

The concentration of anti-pERK should be used at 1:2000 instead of 1:1000 and you should also use 50 ug of protein