Certainly, here is a general protocol for performing western blot analysis on Jurkat cells:

Materials:

• Jurkat cells (cultured and harvested)
• PBS
• RIPA buffer
• Protease inhibitors
• SDS-PAGE gel
• Transfer buffer (such as Towbin buffer)
• Nitrocellulose membrane
• Blocking solution (such as 5% non-fat milk in TBST)
• Primary antibody
• Secondary antibody conjugated to horseradish peroxidase (HRP)
• ECL substrate

Steps:

Note: The specific conditions for each step may need to be optimized based on the protein of interest and the antibody used.

1. Increase your protein quantity/concentration.

2. uneven bands could be charge issue (depending on how you do you run gels). You could let the gel run slowly for 15min and speed up.

3. type of gels like what percent

4. ECL: if bands faint, you can increase the exposure time. But not for a long time.

5. Increase primary antibodies concentration.

6. how many times have you froze/thawed your samples (can degrade your protein)

Do these problems happen to the protein of your interest or all proteins tested, including the internal control?

Hi Nada Mohamady

Rohit S Ranabhat's reply is very informative and covers what you can do experimentally to address problem points 1 and 2, they can also be reduced/overcome by using good analysis software.

You can get a free trial of our Phoretix 1D software here - https://totallab.com/products/phoretix-1d/ which will give you the ability to edit your images post capture and use things like heatmaps to visualise even faint bands helping you solve problem 1).

To help with problem 2, our software will allow you to quantify even wonky/bent bands accurately so that becomes less of a problem, helping you get results where you previously you thought you couldn't and saving you time by not having to repeat your experiment.

I agree to RS Rhanabat's suggestions.

I used to load 50 or up to 150 microgram protein extract for Jurkat cells per well.

In addition, according to the MW of the protein you want to detect, you may modify your transfer conditions.

For example, I was using 100V for 1-2 h to detect a high MW protein (200kDa).

Protease AND phosphatase inhibitors in the protein extraction buffer are also essential. See my protocol in Mourali et al, Mol Cell Biol 2006,26(16),6209. Good luck!