Problem with antibodies - (Nov/06/2006 )
Hi everybody, I am doing dot blots and the primary antibodies do not bind to my protein, what could be the reason? Secondary antibodies (with horseraddish peroxidase) work. Help, im new at it. Does it mean, that the antibodies are wrong?
Thanks for any advice.
Hi, please tell us a bit more about the conditions for your blots, the antibodies you use, the buffers, the time you incubate, how you blot etc. We can tell you more then.
yes tell a little more.
if you were blocking with milk, try to change for BSA, it blocks less.
if you were blocking with milk, try to change for BSA, it blocks less.
So, it continues: my dot blots are a bit inverse, where the primary antibody and metallothionein was, is a white dot
, its hen antibody against metallothionein, secondary is rabbit against hen immunoglobulin conjugated with horseraddish peroxidase. I use PBS and 5% milk, I tried different dilutions and it still doesnt work. And when I try the same process with horseraddish peroxidase, no dot (neither white nor brown) is detected. Is it possible, that the epitopes for secondary antibody are blocked, so the secondary antibody can not bind? yes tell a little more.
if you were blocking with milk, try to change for BSA, it blocks less.
So, it continues: my dot blots are a bit inverse, where the primary antibody and metallothionein was, is a white dot
, its hen antibody against metallothionein, secondary is rabbit against hen immunoglobulin conjugated with horseraddish peroxidase. I use PBS and 5% milk, I tried different dilutions and it still doesnt work. And when I try the same process with horseraddish peroxidase, no dot (neither white nor brown) is detected. Is it possible, that the epitopes for secondary antibody are blocked, so the secondary antibody can not bind?did you make a row of different concentrations of your antigen (metallothionin) as dots? your reported inverse effect may happen if there is too much protein in a dot...
yes tell a little more.
if you were blocking with milk, try to change for BSA, it blocks less.
So, it continues: my dot blots are a bit inverse, where the primary antibody and metallothionein was, is a white dot
, its hen antibody against metallothionein, secondary is rabbit against hen immunoglobulin conjugated with horseraddish peroxidase. I use PBS and 5% milk, I tried different dilutions and it still doesnt work. And when I try the same process with horseraddish peroxidase, no dot (neither white nor brown) is detected. Is it possible, that the epitopes for secondary antibody are blocked, so the secondary antibody can not bind?did you may a row of different concentrations of your antigen (metallothionin) as dots? your reported inverse effect may happen if there is too much protein in a dot...
Naive suggestions .... but may help.
1... what is the protein conc of your lysates?
2... perhaps change the primary antibody. If you're using a commercially available primary, try changing the company.
Hope it works.
what are the concentrations of the antibodies used (for primary and secondary)?
who is the provider of the secondary?
what is the concentration of your sample?
If your membrane is black, maybe you are using too much secondary.
did you try different concentrations of secondary while dotting the secondary antibody?
Did you try different concentrations of secondary on a western-blot on your sample, without the primary antibody to determiine to lowest dilution that doesn't give background?
who is the provider of the secondary?
what is the concentration of your sample?
If your membrane is black, maybe you are using too much secondary.
did you try different concentrations of secondary while dotting the secondary antibody?
Did you try different concentrations of secondary on a western-blot on your sample, without the primary antibody to determiine to lowest dilution that doesn't give background?
My membrane isnt black, its only colored, with the optimizing I begin till it works
I tried to dot the different dilutions of secondary, I tried different concentrations of antigen (original was 1 mg/ml, i diluted it ten and hundred times), and different dilutions of both primary and secondary and still is a white dot on the place of antigen and primary.... I had once a negative western-blot, when I was beginner. the membrane was colored, and the bands were still white, but it was all bands, and not only specific bands.
I was preparing the PBS plus milk in advance, and eventually the buffer became slightly acidic due to contamination.
then I switched to Tris buffer and always prepared the milk extemporously, and I never had this problem anymore.
if you get an inverse dot [or band in a western blot] try loading less cell lysate/protein or using less primary