5 replies to this topic

[than4]

We have a new student in our lab who is running some tissue samples on a WB.
She has done a BCA assay, loads the same amount of protein and probes for her protein of interest.  She then reprobes the blots for actin as a loading control.
The actin bands come up fine, but she is concerned that they are not dark enough and should be stronger.

We use a primary antibody from Santa-Cruz, a mouse secondary from Promega, ECL from Pierce and look on the Biorad Chemidoc.

How strong should the actin band be?  Is she right and it should be darker?  If so, what might the problem be?

Thanks

[Aris]

than4, on May 25 2010, 08:47 PM, said:

We have a new student in our lab who is running some tissue samples on a WB.
She has done a BCA assay, loads the same amount of protein and probes for her protein of interest.  She then reprobes the blots for actin as a loading control.
The actin bands come up fine, but she is concerned that they are not dark enough and should be stronger.

We use a primary antibody from Santa-Cruz, a mouse secondary from Promega, ECL from Pierce and look on the Biorad Chemidoc.

How strong should the actin band be?  Is she right and it should be darker?  If so, what might the problem be?

Thanks

I dont recommend stripping and reprobing.
Cut the membrane and test for different Abs on the same time.
Densitometry should be done at non saturating exposures. If it is black it can not get any blacker
Please upload an image, that will help

Cheers

[bob1]

than4, on May 25 2010, 07:47 PM, said:

How strong should the actin band be?  Is she right and it should be darker?  If so, what might the problem be?

This is a bit of a "how long is a piece of string" sort of question.  They should be dark, but not over exposed is the answer.

Aris, on May 25 2010, 08:39 PM, said:

I dont recommend stripping and reprobing.
Cut the membrane and test for different Abs on the same time.
Densitometry should be done at non saturating exposures. If it is black it can not get any blacker
Please upload an image, that will help

Cheers

If the proteins of interest are of different sizes to Actin, it is perfectly valid not to strip the membrane and just probe for Actin as you would normally.  If your protein of interest uses a different species antibody, you can even mix the two primaries and the secondaries.

IMHO - densitometry = not a good method.

[than4]

I think the problem is that she thinks the actin band should be much darker than that of her protein of interest and is therefore concerned that a) the band is not actin and/or the antibody is not very good.

The antibodies are two very different sizes and two different species which is why I suggested doing it together or sequentially without stripping.  I also suggested that she cut the membrane to see the intensity of the actin band without the original antibody.

However, she has decided that tublin might be a better control..........

[Aris]

than4, on May 26 2010, 07:17 PM, said:

I think the problem is that she thinks the actin band should be much darker than that of her protein of interest and is therefore concerned that a) the band is not actin and/or the antibody is not very good.

The antibodies are two very different sizes and two different species which is why I suggested doing it together or sequentially without stripping.  I also suggested that she cut the membrane to see the intensity of the actin band without the original antibody.

However, she has decided that tublin might be a better control..........

Well, she can choose whatever control she likes but the criteria should not be the  intesnity of the band but the fact that the expression of the control protein is not changing by any treatment. A preliminary experiments shoudl help define which is the best control marker.

[Aris]

bob1, on May 26 2010, 05:26 PM, said:

than4, on May 25 2010, 07:47 PM, said:

How strong should the actin band be?  Is she right and it should be darker?  If so, what might the problem be?

This is a bit of a "how long is a piece of string" sort of question.  They should be dark, but not over exposed is the answer.

Aris, on May 25 2010, 08:39 PM, said:

I dont recommend stripping and reprobing.
Cut the membrane and test for different Abs on the same time.
Densitometry should be done at non saturating exposures. If it is black it can not get any blacker
Please upload an image, that will help

Cheers

If the proteins of interest are of different sizes to Actin, it is perfectly valid not to strip the membrane and just probe for Actin as you would normally.  If your protein of interest uses a different species antibody, you can even mix the two primaries and the secondaries.

IMHO - densitometry = not a good method.

Agree

Cheers