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Confusing ELISA results - not scaling with concentration


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#1 bdg25

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Posted 12 November 2014 - 11:29 AM

Hi All,

I am working on an ELISA for a protein found in HeLa cells.  It has a MW of about 12 KD.  I had the recombinant protein made as well and am testing both cell lysate and the recombinant protein in an ELISA format.  I am diluting both of them in coating buffer + roche complete protease inhibitors.

 

For the recombinant protein I just made concentrations of 25 ng/mL, 10 ng/mL, 1 ng/mL 100 pg/mL and 10 pg/mL.

 

For HeLa lysate I was worried I was preparing it wrong due to bad result before so bought some from Ray Biotech (

I diluted it 1:10, 1:20, 1:40, 1:150, and 1:300

 

The problem is that I got decent results for the recombinant protein but for the lysate I can not make any sense of them. The negative control showed a consistently low signal (control samples consisted of just coating buffer + protease inhibitors) but regardless of the dilution of cell lysate, the signal was identical.  

 

I don't understand what would cause this because the background is very low in the negative control, how is it equally positive (and very positive at that) regardless of the lysate concentration?

 

 

Protocol overall:

 

Place 100 uL sample in 96-well corning EIA plate overnight at 4C.

Next day, wash 3x with PBST, 5 minutes each

Apply 300 uL blocking buffer (5% BSA in PBST) for 2 hours with gentle rocking

Wash 3x with PBST, 5 minutes each

Apply 100 uL of 2 ug/mL mouse anti-target monoclonal antibody for 1 hour at RT with gentle rockiing

Wash 3x with PBST, 5 minutes each

Apply 100 uL of anti-mouse IgG at 2 ug/mL (abcam recommendation for this secondary antibody)

Wash 6x with PBST, 3 minutes each

Apply 100 uL TMB for 15 minutes

absorbance at 450 nm

 

 

Can anyone help me make sense of this? I just really do not understand the signal for the lysate

 

 

 

 



#2 BioMiha

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Posted 24 November 2014 - 02:29 PM

Hi bdg25,

 

Even though you can in theory do it this way, I wouldn't actually coat the HeLa lysate on an ELISA plate. Antigen ELISAs are usually done in a sandwich format, with mAb "A" coated on the plate, after which you float the sample or recombinant protein over that and then you detect by adding labelled mAb "B" (e.g. biotinylated + streptavidin-HRP). It makes it much less messy, hence eliminating background noise. 

The other thing I would ask is where you got your detection mAb from. Some vendors are more reputable than others...

 

Hope it helps.






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