Hello,

can you give us more info on what your study material is ?

- Cell line ?

- transfection/overexpression ?

- the antibody you use ?

I didn't quite understood what you said about the detection,

you can't see the protein when staining the gel nor with your antibody in western.

But you see the protein when staining the western blot membrane ?

like with ponceau red ?

Hi Ru Nm

* To detect small molecular weight proteins (less than 15 kDa), it is recommended to use a 15-20% gel.

* Also you can check how the antibody blots appear in its website and its specificity and reactivity accordingly.

Thanks,

I am working on lair2 and how it functions in lupus

we try to detect the protein many times in western blot, the samples were from pateints.

we removed the albumin and IgG by albumin /IgG removal kit.

first we used 12.5% gel to detect the protein by antibody, and that didn't go than we moved to 15% gel and we staind the gel with coomassie blue after the gel electrophoresing the samples but we didn't see it ( we saw faint band and we are not sure if this belong to lair2) .

Ru Nm ok you work on clinical samples.

Do you include a positive control in your western blots ?

Like a cellular extract coming from a cell line known to express your protein of interst ? Or coming from a transfection of a vector overexpressing your protein ?

As Samir pointed out, you could use higher gel percentage to better resolve the band corresponding to your protein but if it is there and expressed at good levels in your samples, even 12% should be able to give you a good signal.

The sensitivity of coomassie staining is really inferior to what you should get with your antibody so I wouldn't rely on that.

How much protein in µg do you charge in your gel ?

I beleive you must be limited by the amount of clinical sample you have ?

Best of luck,

Philippe.

the samples incuded 40 µl of the pateints samples and 10 µl sample buffer (1:5).

is there is another method I can detect the protein in the samples?

Ru Nm I think your best choise is to determine:

first: if your antibody is working

second: the protein concentration of your sample

By the way, do you have a blot image ? Can you detect your control protein ?

Hi Ru Nm,

The protein concentration in the sample is to be detected first. Use 15% gel. Use a ladder. Why do you stain the gel? Transfer it to PVDF membrane (as you said you use Coomassie blue). Ponceau goes well will all membranes and it does not affect the polypeptide. It can be removed completely so that you can proceed with further.

first we try the western blot , we transfer the proteibs to the 0.2µ membrane

and incubate it with the first antibody O.N and the second antibody. we did all the steps of the western blot and we didnt succeed to detect our protein .

it's a secreted protein .

Ru Nm Hi again !

Ho if it is a secreted protein, you may need way more than 40 µL of sample I'm afraid.

What is the total amount of sample you harvest from patients ?

You can try to concentrate your sample prior to western blotting.

thank you I well try