Do I need to do BCA assay to quantify the protein before doing the ELISA? - BCA assay and ELISA (Jan/28/2008 )
Do I need to do BCA assay to quantify the protein before doing the ELISA? I am doing the siRNA knock down experiment. I want to see whether the protein of my interest can also be knocked down. unfortunately there are dimers for my protein on western blot. there are also this protein in the serum of culturing medium. I can not differetiate whether it has been down regulated by western or not. is it possible for me to detect whether the protein is down regulated by ELISA?do I need to load equal protein to ELISA?
many thanks.
ofcourse u need to quantify the protein before ELISA, if u want to compare two conditions.
but i do not understand the problem with WB.
how u see the dimers in the western blotting?
If you have a good set of standards of your protein, you can avoid the protein quantitation before the assay. Also, unless your protein preparation is pure, you'll be measuring whatever else is in the sample.
is int tulip1 supposed to load same quantity of lysate from both transfected and untransfected cells.
good standard is int only help in good quantifiction of protein after transfection? but not for the normalization and/or comparison......
thanks for your reply.
I saw two bands on westen blot. one is around 50kb(dimer), the other band is at 25kb, chemicon did not say it can be used for ELISA, Do I need to buy a new antibody and a separate new standard?
I want to load the samples(medium or cell lysate) from different groups (siRNA treated and scramble control treated), I am wondering whether I need to load equal amout of protein in ELISA or not? from the answer above I suppose I should even if I have a good standard curve? Is there any other method which can take place of troblesome BCA assay? if I only want to compare whether it has been knocked down or not, Do I need to buy a protein to do the standard curve? I felt headache to ask my supervisor to buy the stuff for me as he always says his money has gone.
OK. hear is the deal,
u must load same protein quantity from both conditions, use bradford or 280nm as BCA alternative.
If u want to check protein in soup, use equal volumes (no protein estimation).
regarding the second point, perforom an ELISA with those antibody from chemicon, if u get color then u can use it for ELISA too, after fine tuning...
gud luk
thanks for your reply.
Do you mean if I want to check the protein in soup I don't need to do BCA assay? but there is also possibility that there are no equal total protein after knocking down.
If u want to check protein in soup, use equal volumes (no protein estimation).
s i agree with u, after knocking down ur protein, u will nothave similer protein concentrations. but its u who shud decide which way u want to compare for normalization.
example- if u r knocking down a structural protein then u will have significant reduction in protein quantity compared to total protein.
but ur protein is a regulatory one or avialable very less then u can quantify the protein n normalize it.
ideal situation for normalization after transfection is counting cells, take equal number of cells from both conditions lyse them in equal volumes of lysis buffer and use eqal volumes of lysate for ur ELISA, then u can express the % of reduction with respect to cell number.
FOR supernatant this probelem does not exist, just take equal volumes and perform ELISA. if u donot get convinced ask ur boss whether this way of doing is acceptable or not?
gud luk
many thanks.
Dear tulip1,
Dear tulip1,
First I would like to know , whether your protein is secretory protein or not. If your target protein is not sceretory protein ,wash your cells before doing WB.if your knock down experiment worked ,you wont find or you will see very minute amount of your protein in cell lysate,compared to your controls.
try to do immunoprecipitation .
For your second question about ELISA, it’s difficult to quantitate in indirect ELISA from the culture medium or cell extract. you need to have sandwich ELISA for the quantitative measurement of protein (for lower concentration) .. In WB, you do see dimer formation(for specific protein),stability of dimer depends on the nature of protein also .
All the best for your knock down.
By
SANDWICH
thanks for your advice.
my protein is maily in the extracellular matrix(under and outside the cells), a little was secreted into the medium. I have found mRNA was knocked down. however I can not detect knock down by western. i have done ELISA before, but I have no standard at the moment, may I do ELISA to compare the difference in protein expression between the different group without doing the standard curve at the moment? if it worked I will buy a standard( it is so expensive!)
what do you mean to have sandwich elisa for the quantitative measurement of protein? I know it is a way of accurate quantification of protein. but I am not sure if it is a feasible way to compare the expression without normalization. though I found most of people did not do normalization of equal protein before doing ELISA.
to count the cells and then lyse seems to be a good ideal.but it is a little bit of troublesome, and I found sometimes the counting is not very consistent.[/b]