No signal/bands observed on western blot for ERK signaling?

I ran a western blot using 12% gel and probing for ERK/pERK (cell signaling antibodies). The green is total ERK, the Red is supposed to be phospho-ERK. I see 2 bands below the total ERK that express the red pERK antibody... but this shouldn't be there.. the phosphorylated protein should be around the same size or higher than the non-phosphorylated ERK... anybody else have this problem? What are these bands?

I've begun examining ERK phosphorylation in vivo and previously my antibody for total ERK (Sigma M5670) gives me 2 bands (ERK1/2) in my HEK293 cell line, but now using it for mouse brain samples I'm getting three bands, the third band showing up at around 46 kDa. I have read that there is an isoform, ERK1b,(Yung, Y., Yao, Z., Hanoch, T., & Seger, R. (2000). ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK. Journal of Biological Chemistry, 275(21), 15799-15808.) that is 46 kDa, and wondering if this is the band that I'm seeing and if this is normal.

Thanks,

Tom

I want to know Erk activity in my animal model - sepsis.

I isolated it from liver, homogenized it, separated it by SDS-PAGE, and detected it, but I just found one band for Erk.

Procedure was the following:

1. block for 1h @ room temperature (RT) with 5% BSA

2. 1:1000 Erk or p-Erk (cell signalling) in 5% BSA, for 1h @ RT and O/N @ 4'C

3. 1:5000 2nd Ab, for 1h @ RT

How can I get two bands for Erk?

Because most papers show two bands for Erk, is there a problem that mine just shows one band for Erk?

Hi all,

have you ever realized how many protocols are available for mesoderm differentiation???

Example:

CHIR+ACTIVIN in E6 medium,

CHIR+MIM

BMP+Ascorbic Acid,

RPMI+Ascorbic Acid...

What are the parameters to select the best protocol for mesoderm differentiation?

I would really like some suggestions!

Thanks

Hi all, I am trying to extract RNA from 12 well plate and so far the yield obtained from trizol/chloroform protocol is OK.

After that I tried to do DNAase step (using NEB DNAse I) but I found that it does not allow cDNA transcription.

I don't know if it was the inhibition step 70°C x 10min of DNAase activity or smth else.

Do you have any similar experience? if yes, any trick to share with me that can help to do the DNAase step without inhibiting the reaction downstream? Thanks a lot

Hello. Hope everyone is doing well. i purified my protein with the Q column (ion exchange column). The first time everything was ok but when I tried for the second-third time i got lots of DNA contamination along my target protein. My protein includes no tag and also tried to change the protein buffer from PBS to PBS(2M NaCl) using a gel filtration column and again change to normal PBS. I lost half of my protein and the left one also was the same. please let me know if you have any suggestions. thanks

Hi,

We will like to receive advice to improve RNA quality and quantity.

After many trials and calibration, we get a protocol that gives better RNA quality and enough quantity for downstream application (cDNA library preparation for RNA seq).

We work with mouth epithelial cells, and because of the presence of many enzymes in the saliva, it's very difficult to obtain non-degraded RNA (not too much degraded) and enough material.

Today, we have a protocol that gives an RNA output with RIN ~4.5 (tapestation), and ~260 ug/ml (Qubit).

Our Protocol:

Using two swabs (Qiagen) we take mouth cells and throw swabs into a tri-reagent/trizol solution. We load this mixture on a shredder (we try without the shredder step, but the RNA was with lower RIN ~2.5 and 10 times less quantity). Then we load the solution on a column and follow the protocol from the kit direct-zol RNA prep, Zymo.

We try other kits:

-RNeasy (plus) micro from Qiagen, RIN between 1.8 - 2.5 and 1000 - 1500 ug/ml.

-RNeasy micro (and mini) from Qiagen, RIN between 1.8 - 2.5 and 1000 - 1500 ug/ml (similar results).

-Quick RNA prep from Zymo, RIN about 1 and ~500 ug/ml.

We begin with the RNeasy with 1 swab and receive RNA with half concentration.

We also try RNAlatter (Qiagen and Sigma), but the extractions didn't give good results.

Thanks

Laure