Unfortunately, w/o ampholyte you cannot establish the desired pH gradient on IPG strips. Observed 1st dimension of protein separation will not reflect the accurate isoelectric points and the following PAGE will not give reliable results. This also affects the resolution, particularly for proteins having closer pI. IPG strips cannot serve pH linearity without these carriers in your rehydration buffers.

Thank you for the detailed answer.

Hi Ehsan,

I dont think that you can do a Bradford Assay with your sample in rehydration buffer. Urea at the required concentration will impact the Bradford assay. Please get info from one of our publications on this topic (attached).

It is also not true that IPG strips will not work without ampholines. They do not require ampholines for the gradient since the gradient in the IPG (Immobilized pH Gradient) strip is immobilised during the polymerisation of the gel by using acrylamide derivates with different pH to form the gradient.

However, the ampholines can be quite helpful to keep the proteins in a solubilzed form. Since ampholines are available at a quite high concentration you should be able to add a few microliters to your sample before doing the rehydration.

Best,

Murat

Hi there, this is the reason this kit (see below) was invented. It is compatible with almost any buffer, so a good alternative to Bradford in your case.

https://www.gbiosciences.com/Protein-Research/NI-Non-Interfering-Protein-Assay

I hope it helps!

Hi,

https://www.thermofisher.com/order/catalog/product/ZM0022

(Carrier Ampholytes are small, soluble molecules with both positive and negative charge groups. They sort based on their isoelectric points in an electric field, thereby establishing a pH gradient in solution or contributing to the gradient when used with IPG strips. Carrier ampholytes help stabilize the pH gradient and current in IPG strips and aid in protein solubility, resulting in reproducible IEF resolution.)

https://www.bio-rad.com/en-tr/product/ampholytes?ID=37d2370e-0a3f-4fdc-8c4d-de1e2f69f6eb

(carrier ampholytes are blended to give a complete range of isoelectric points for reproducible, linear pH gradients.

• Wide-range ampholytes have a working pH range of 3.5–9.5
• Narrow-range ampholytes make it possible to further resolve many components of complex mixtures within a narrow pH range and provide greater separation of proteins of slightly differing pI values)

reminder;

w/o ampholyte you cannot establish the desired pH gradient on IPG strips. protein separation will not reflect the accurate isoelectric points and the following PAGE will not give reliable results. This also affects the resolution, particularly for proteins having closer pI. IPG strips cannot serve pH linearity without these carriers in your rehydration buffers.

Hi Ismail,

I dont know where you have this last info from please provide a reference for your statement after "reminder" and in your initial statement.

This is complete nonsense. I have done thousands of 2D separations and some of them also without supporting ampholines and again with good comparable results.

Maybe you are using the wrong IPG strips?

And again the ampholines should support the solubility of the proteins but are not required for the formation of a gradient for IPG strips as they are required in the original 2D protocol (O'Farell or Klose).

Best,

Murat

I would like to isolate transferrin isoforms (for CDG diagnosis) using IPG strips.

Do you have any protocol for IEF by strips and 2D? I also want to know the treatment protocol for preparing serum.

I value the insights and guidance you provide