Hi all,
I did prepared a sample for mass spec, before drying the sample I took 5% to run it on an SDS gel. I have to add Lameli samplle buffer. I am afraid that my protein would agrgate so I am thinking of adding Urea to a final concentration of 4M.
Do any of you have any expireance, is 4M Urea sufficient?
Any other sugestions?
Guy

hi Guy, most people use 8M, I am using 6M, and it works fine.

Hi again
Just got the results,
4M Urea is very good for my protein CFTR.
Got nice resulotion.
I will deposit teh IP protocol with Basic elution in the comming next days.
Guy

Hi,
Here is the western
Bolted with anti CFTR
the first two are just lysate with 10Xlameli sample buffer. the second set:1st is sample with lamely sample buffer and 4M Urea final concentration. As you can see this lane is the same as the first lane in the first set.
the other are IP of CFTR

Anotjer Question,
Do any one used DSP cross linker?
If so can you cleave it using DTT in AMmonium Hydroxide?
Guy

Got the answer from a mass spec expert, he sais that you can add the DTT100mM to the Ammonium Hydroxide the elute the Ab protein complex and then alklyse with 2oomM Iodoacetamide.
Did it today and will send it to mass spec later on this week.
Guy

Got the results today and the sample that contained DTT didnt yield any results.
Today I am preparing a new sample which will contain DTT but no Iodoacetmide.
Guy