Hi Shoxan Abdullah

Before, going into the problem associated with western blot, you must discuss the whole process, what you have followed. So, all possible reasons could be explored rather just pointing antibody, lets begins with

1. Do Protein concentration was good enough?

2. What was the SDS Gel percentage? The molecular weight of the protein?

2. What was the transfer protocol/method: Voltage, current, and duration?

3. Have you tried Ponceau S staining?

4. Have you done Antibody titration to get optimal concentration/dilution

5. Very important how was your housekeeping protein expression?

6. Any Positive or Negative control used?

With such basic details, every member can help you to troubleshoot.

Regards

Saurabh

In western blot you can face many problem, please check the following things you are usin.

1. First check the pH of TBST, if the pH is acidic it means they wash your protein too or denature from membrane when you washing it.

2. Check your primary antibody is the are working to attach with protein. or prepare new one.

3. Check secondary antibody they are specific or not with the antibody you have purchased, means the primary antibody need antigoat or mouse etc. Also check they are working well with another antibidy.

4. Use cool transfer buffer.

5. Check the time of transfer your protein need. means if have high KDA will need more time for transfer or if have low KDa will need less time.

Hope it will help.

You need to do this systematically:

• Is your protein of interest present in the sample (spot blot)
• Does the electrophoresis work correctly (stain with RuBPS or similar)
• Does the transfer work (staining the gel should be negative, staining of the blot with Ponceau should be positive for protein bands)
• Does the primary antibody recognise the denatured antigen (some ab work only with native target. You could try a spot blot with both native and SDS/bME denatured samples)
• Do the secondary antibody and the coupling enzyme work (spot blot with strongly diluted secondary ab as sample)

thank you for your answer

the protein of interest was in sample spot

and my electrophoresis worked and I sow band in it Engelbert Buxbaum

but the rest I am not sure I will change my primary antibody because I feel its not suitable for my interest but how I can prove the my secondary antibody and coupling enzyme work there is any way to prove that please

my gel was 18% and I did Simi-dry transfer to membrane Bashir Ahmad and my protein was worked on sds pages and I detected band on gel but not in membrane

Do you know if the mAb you used is intended for western blot? Typically, polyclonal antibodies work better.

Please recheck the following:

1. Concentration of your protein lysate ( get your standards right, ALWAYS ADD YOUR BUFFER BLANK and calculate unknown from it, make sure to add the required dilution factors). Buffer blank is really important because your protein concentration will be derived from there only!

2. Do a commassie on your gel to visually see protein profile ( to me seeing is believing).

3. Run your samples on gel (add a positive control without fail), transfer and then stain with ponceau. That should again tell your efficiency of transfer.

4. Block for 1 h RT with milk

5. Use a good quality antibody and incubate overnight. Santacruz, CST works good even with 1:1000 dilution. You could put your primary in 5% BSA/TBST. It works best for me.

6. After secondary incubation, wash blots and develop. First see if your positive control is showing expression. If that comes up then you don't have to worry

much.

7. Probe a loading control too.

Good luck!

The 18% gel is suitable for low molecular weight proteins like 15KDa or below. So I am not sure what is the MW weight of your protein. If your protein you have detected on gel and not transfer, so maybe you place on wrong side of membrane or problem in antibody. Shoxan Abdullah

• Shoxan Abdullah : To prove that your secondary ab can recognise the primary, do a dot blot with a very diluted solution of the primary. You can also spot diluted secondary to check that the coupling enzyme works (and your reagents too!)
• Did you stain the membrane with Ponceau Red (doi:10.1016/0003-2697(86)90263-0) to demonstrate protein bands? Ponceau is reversible, the stain is removed during the blocking step. India ink (doi:10.1016/0003-2697(83)90237-3) is more sensitive, but irreversible.
• Are you sure that you had the membrane on the right side of the gel during blotting? SDS is negatively charged, so the membrane must be on the positive (red) side.