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western blotting troubleshooting - (Feb/15/2013 )


I have problem with my western blot nitrocellulose membrane. I used first primary antibody: rabbit anti GAPDH and I had a good signal. after this detection the membrane was washed 5 time at 5 min with PBS with 0,2% tween and I used a new antibody. And the step was:

1. blocking 30min again in 5% Non fat dry milk
2. a second primary antibody : rabbit anti ID3 for 2h at RT
3. wash 5 time at 5 min with PBs/tween
4. secondary antibody goat anti rabbit biotin for 2h at rt
5. wash
6. streptavidin POD (ultrasensitive - sigma) 1:1000
7. wash
8. ECL detection with roti-lumin (roth) 1ml for 4 min on the membrane and exposure time 5 min.

I did not have signal for second primary antibody, after 5 min ......even no background. This is strange because for the firs one I had a good signal in the same condition. What can be the problem?

Thank you


Did you strip your membrane? What are the sizes of your proteins you are identifying? Why didn't you add both primaries simultaneously? Are you positive your Abs are working properly. Did you increase your exposure time after you realized that 5minutes did not work? Is it possible that you had such a small ug of protein on your gel that a longer exposure (<2hr) was needed to visualize the protein?

I would take a protein sample that you know is expressing high amounts of your proteins and run a titration for them. See what ug of protein gives you the best result for your Ab.

Make sure you minimize the amount of freeze/thaw cycles on your Ab. Depending on the amount of sodium azide (or other stabilizer) in your Ab, it will affect the longevity of your Ab.

Also, be mindful about your nitrocellulose membrane (I'm sure you know this). Be gentle with it. Make sure your protein transfer side is always up. Nitrocellulose is more susceptible to losing protein as the pores within the membrane do not close around the protein following transfer (methanol in PVDF activates and closes pores around proteins).

Good luck!

What % of milk are you using for your primary and secondary Abs? Is it 5%, like your blocking concentration?


No, I did not strip the membrane. The size of protein is 10KDa. I am using 5% milk for both primary and secondary antb.
I did not increased the exposure time. I dont know if POD ECL substrate is still active after 10 min (this is the recomandation of roti lumin). The membrane was with de right face up and the antibody is ok because it work in immunohistochemistry. The substrate should be ok also.
But it is possible to did not see the background?
I din not add the primary antibody together because they are both rabbit. But this is a good ideea for future.
I will ty to use other antibody today, to see if here is signal or not and I will be back with result.

Thank you for your answer it is very useful for me.