5 replies to this topic

[hanze]

Hello

I´m working with a Phospho-Elk1 (Ser383) specific Antibody (Santa Cruz) but the detection of the right band seems to be a hard piece of work since there are some bands at the expected size. I already tried the appropriate blocking peptide without any success.
Is nuclear fractioning absolutely necessary for proper detection of pElk1 on Western Blot? (HeLa cells). Somebody got a good protocol?
Could it also be possible that the phosphorylated protein runs even further than the unphosphorylated one? Is it possible at all that you can see a migration difference between pElk1 and Elk1 in Western Blot?

Hope somebody can help me to identify the right band.

[Gourav Mishra]

Hi !

cell signaling phospho-Elk1 antibody (cat#9181) give a clean 62 kDa band upon using their protocol, you may try.

Ruchi's protocol is okay . . . . .. but Elk1 doesnt express in all cells . . ..  . if your cells are proliferating . . . you should get the band.

Gourav Mishra

[polyfractal]

You will not be able to resolve by western blot the difference in pELK1 and ELK1, the weight added by the phosphate is miniscule.  Make sure you are blocking your blot membrane with BSA instead of dry milk, as casein (in the milk) is a phospho protein and can lead to nonspecific bands when probing for phospho-proteins.  It is also recommended to blot overnight at 4 degrees instead of an hour at room temp.

Perhaps try running serum starved cell lysate next to regular lysate and probe for pELK?  Since pELK is upregulated by serum and growth factors, you should probably see a difference in the band intensity between serum starved and serum.

Lastly, try the Cell Signaling antibody, they make superb antibodies.  In my experience, Santa Cruz antibodies are very hit or miss, more often missing.

Edited by polyfractal, 22 July 2009 - 09:04 PM.

[SCBT Technical Support]

Hi there,

This is Technical Support at Santa Cruz responding.

If you are having troubles with one of our products (in this case, p-Elk1 (Ser383)), remember to always contact us. We are here for our customers and want you to have success using the products purchased from us. We do warranty our products and their performance, so please do contact us if you were unable to get results using the antibody.

We are always concerned when a customer has troubles with one of our products, so if you haven't done so already, please do contact us at 1-800-457-3801 (or scbt@scbt.com).

Thank you & good luck!

Technical Service
Santa Cruz Biotechnology, www.scbt.com
Email: scbt@scbt.com
Phone: +1-831-457-3800 ext. 2 or toll free 1-800-457-3801
Fax: +1-831-457-6013

[Dorkaitz]

Hi!
I'm working with a phospo-ELk1 (Ser383) antibody from Cell Signaling (#9181). You say that this antibody gives a clean 62KDa band upon using their protocol, but I do not detect any band. I started the western blot from a whole NIH 3T3 protein extract. I loaded 40 micrograms of protein extract and I incubate the antibody (1:1000 dilution) overnight at 4ºC with gentle shaking in 5% milk. I wonder whether you will be so kind to answer some doubts I have about the western:
What type of cells do you use? Do you start the western from a whole extract or a nuclear extract? Shall I incubate the antibody with BSA instead of milk? Shall I use a higher concentration?
I am also thinking about buying another antibody. Does anyone know if the Santa Cruz antibody phospho-Elk1 antibody (sc-8406) works well in these kinds of cells?
THank you very much in advance

[mdfenko]

i would consider using bsa instead of milk. milk contains phosphoproteins (casein) which may be depleting the antibody.