protein quatitation before ELISA

9 posts / 0 new
Last post
nani
nani's picture
protein quatitation before ELISA

Hi every body,
I'm going to perform ELISA to detect phosphorylation of EphB4 receptor tyrosine kinasae after ligand stimulation.
In the ELISA  kit manual, it is suggested to quantitate sample protein concentration by a total protein assay before running ELISA. To do so, I should quantitate the total protein from supernatante of  lysed cells &
In the same manual it is suggested to use lysates immidiately for ELISA.
My question is: Quantifying total protein may take time and thus I won't be able to start ELISA immidiately, moreover, from different existing protocols for total protein quantitation, I don't know which one is appropriate for this situation.
I'm stuck in a terrible situation and need to solve this problem immidiately
SOS please

Sami Tuomivaara
Sami Tuomivaara's picture
farnazb,

farnazb,

Immediately here means: "Do not put your protein in fridge/benchtop for overnight".
Protein assay takes anywhere from few minutes to half an hour depending on the assay, it is fast enough. Remember that the ELISA incubations take usually even longer, so short time wait is ok. I suppose you have protease and phosphatase inhibitors in your lysis buffer, they are a must of course.

Suitable protein assay depends on the reagents you have in the lysis buffer because every assay is interfered by different reagents... Here's a comparison from Pierce, you can find more information by googling

Cheers,

nani
nani's picture
Hi Suola,

Hi Suola,
Thanks for your immidiate hint.
you mean i can leave my cell lysate in room tempareture and do the protein assay?( if my final choice is to be Lowry method, it will take time to perform it)
My lysis buffer is consist of :
1% NP-40,20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2mM EDTA, 1mM activated sodium orthovanadate (Na3VO4), 10 mcg/ml Aprotinin, 10 mcg/ml leupeptin.
Thus I have both chelator (EDTA) and detergent (NP-40) in this buffer both of which are incompatible for Bradford & Lowry respectively.
 My other question is which method ( Lowry or Bradford) is more sensitive and/or convinient for this case?
it's very kind of you to give me your idea.

Sami Tuomivaara
Sami Tuomivaara's picture
farnazb,

farnazb,

It is advisable to keep your lysate and protein solutions on ice...

Bicinchonic acid assay is forgiving for both NP4- and EDTA at the concentrations you use.

Cheers,

nani
nani's picture
Thanks but unfortunately I

Thanks but unfortunately I don't think it would be possible for my supervisor to provide me BCA materials at the moment & only bradford or lowry is accessible now. Do you have any idea in choosing one of these two or you think it's better to stop working and get BCA? 

Chin Fen Teo
Chin Fen Teo's picture
 Hi Farnazb, 

 Hi Farnazb, 

While as Suola mentioned that BCA may be the best method to go with samples containing detergent, it is not impossible for you to stick with Bradford assay with the lysis buffer that you are using- In fact, I routinely use Bradford assay for my samples containing 1%NP40+0.1% SDS+EDTA (and sometime even sodium deoxycholate).

As long as your sample to dye ratio is able to keep the concentration of NP-40 per se within the compatible range, you should be fine. For instance, when I am doing Bradford assay in microplate format, I alway use 1 ul of sample in total of 200 ul reaction, as adding more than 1ul of sample (thus higher content of NP40) will totally turn the color bright blue.  Whereas for the case when the classical cuvette is used, then I usually use no more than 5 ul  sample per 1ml of reaction.

Of course, the concentration of  your samples is another factor that you have to consider, as BCA is more sensitive to Bradford (and Lowry). If you have enough amount of proteins, it really doesn't matter...

Good luck. 

samm
samm's picture
I've used Bradford at 1:250

I've used Bradford at 1:250 (microplate reader) using a similar buffer to yours, so given both pippuri and my experience with it, there is no reason for you to stop your work. However, BCA&nbsp;does work better across a range of sample concentrations and compositions - I&nbsp;recommend the Pierce kit (or eq) that has everything to get you going.<br /> Worst case scenario (if you don't have either eragent available now), you can try a protein measurement at 205 nm, though standardizing that is very very hard (each protein has an ever so different std curve, and mixtures of proteins in lysates will only result in very approximate quantification).<br /> Suola, are you aware of any alternatives to BCA that still maintain 'inertness'?<br />

Sami Tuomivaara
Sami Tuomivaara's picture
samm: BCA seems to be the

samm: BCA seems to be the bests choice, after that I'm out of ideas...

Farnazb: Maybe you just have to go ahead and measure protein concentration with colorimetric assay or as samm said, with uv absorbance.

Approximate quantification is better than none at all. Besides, absolute protein amount in this case is not as important as the fact that you load equal amounts from different samples to the gel. So go ahead and see if the background is reasonably low. If you get a value from your measurement, at least the background is the same in all samples that are in the same buffer...

Also, you can later do another western with adjusted loaded volume if the first time you load too much or too little.

Cheers,

nani
nani's picture
Thanks to all you wonderfull

Thanks to all you wonderfull scientists.
 So much thanks for your generous help. Your sense of reponsibility & cooperation to share your valuable experinces is really admiring & encouraging.
I think i have to choose Bradford cause I already have the reagents in the lab. As I understood the amount of sample to dye is important . from your experience, do you know any written protocol that you follow for microBradford ( with suitable sample amount required).
.