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Western-No bands for sample protein but loading control works

western blotprotein protein extraction

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5 replies to this topic

#1 HawkeyeGrad

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Posted 12 July 2018 - 09:31 AM

Hi All,

I've been trying to troubleshoot this western blot for weeks and I need help. I'm blotting for my protein of interest, LOX, and am using GAPDH as loading control. I am able to detect the loading control always on the blot but can't detect the protein of interest. I've tried several different conditions including adding up to 150ug of protein and still no dice.

 

1. Extract protein in RIPA with protease and phosphatase inhibitors by sonication and incubation on ice for 30mins. (Also tried Protein extraction kit from Full Moon)

2. Load 40-50ug protein

3. 10% SDS page

4. Transferred to PVDF membrane for 1.5 hours

5. block for 2 hrs in 5% milk TBST

6. 1:1000 primary co-incubation (1:1000 GPADH) overnight at 4C (also tried 1:500) in Odyssey buffer

7. wash 3x 5min in TBST

8. 1:5000 secondary incubation for 1 hr at RT in Odyssey buffer with LiCor secondary antibodies

9. wash 3x 5 min in TBST

10: Image with Bio-Rad Chemidoc

 

I also run a positive control protein sample to the blot (from the same company as antibody) and the antibody recognizes this positive control and I see bands only in this lane.

 

My loading control always shows up as a nice band but I can't detect my protein of interest. I was thinking the antibody may just be bad but the reviews online and the datasheet kind of argue that the antibody should be pretty good for western blot....

 

ANY help/suggestions would be greatly appreciated!



#2 mdfenko

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Posted 12 July 2018 - 09:59 AM

have you tried putting the positive control through the same extraction process as the sample (ie add the positive control to the extraction sample)? you may be losing the protein during extraction.


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#3 HawkeyeGrad

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Posted 12 July 2018 - 12:05 PM

The positive control is already denatured and purified so I'm not sure if subjecting it to the same extraction process would work. But I will try that and see. Would I still be able to detect loading control GAPDH though if I'm losing protein during extraction?



#4 mdfenko

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Posted 12 July 2018 - 12:44 PM

the gapdh may be in more abundance and/or more resistant to proteolysis.

 

is the positive control denatured in sds-page sample buffer?


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#5 HawkeyeGrad

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Posted 12 July 2018 - 12:47 PM

Yes the positive control is supplied read-to-use and denatured in SDS-page buffer.



#6 Artemis2007

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Posted 13 July 2018 - 12:02 PM

I would try it using a lower blocking conditions than the Odyssey buffer for all your incubations - e.g., BSA and/or milk 1-5%. Then use HRP-linked secondary with ECL to visualize.  Some proteins are hard to see in the Odyssey system, in my experience. The purified positive control suggests the antibody works but you might need to optimize for your samples. Is it possible your POI is not highly expressed in your cells? Maybe you could induce expression in vitro as another positive control.   Also you could check for published citations and see how others may have prepared the lysates. 







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