The problem could be with the anti-His antibody. Some of the commercial ones don't work very well on Western blots. Try a different brand, or use an antibody specific for the protein rather than the tag.

By the way, your SDS-PAGE looks pretty bad. You may want to think about what is causing that: is it the gel itself, or the sample composition?

I agree with Adam, some His-Ab do not work good especially when little protein amount there.

But regarding your purification , maybe you describe better and can get help to optimize htat procol, as it looks as if you loos protein during wahing already , no ?

How are you wahing and eluting ? with Imidazol?

How are you blotting ?

Thanks Dear Adam, the pAb anti-His is quite old but at the same time it worked with control +ve, unfortunately, we don't have a specific Ab against our r-protein. 

I agree the SDS is quite strange this time, may be because of the high protein conc and sample stickiness in the most right lanes.

Hi Vanessa, 

why do you think that the washing is not good enough, If you mean the same molecular weight band in the washed fraction lane, may be because of the column capacity matter, actually I am using the 1 ml anti-His column to purify the r-protein from 50 ml culture broth; yes I am using Imidazole 5mM and for binding and washing and higher for elution. I blot with pressure using a heavy weight to transfer the protein from the gel to the membrane: no electricity is involve as usual.

Hi Haitham,

To a naive observer, can you explain why you are using physical blotting of the samples onto the Western membrane rather than electrical current to mediate the transfer?  This method sounds more similar to that used for Southern / Northern blotting and is not a protocol I am familiar with for protein gels, so it is interesting to hear about this technically.

One thought that comes to mind to eliminate the transfer method as an issue - could you try undertaking spot blots to determine whether your antibody is able to pick up protein that is directly spotted on the membrane compared with a control sample from a well that does not contain the protein (ie comparing positive control with elution samples with late wash samples pre-elution etc.).

Can you also describe other conditions for the column purification - the composition of the culture broth / binding buffer, wash & elution buffer pH / salt concentration etc for the various steps?  Which 1ml His-column type are you using?  One that is designed for application of crude lysates or something less tolerant?

I was also wondering whether there is any scope to use (for instance) ion exchange resin as a crude capture / cleanup method prior to using the His column based on the parameters of the protein (pI etc).

This is certainly an interesting question and one well worth discussing.

Hi Robin,

Thanks for joining this discussion,

1- Simply, because they used to use the physical blotting for long time here in this lab and it works very well with most of proteins, and they argue that they can produce more than one blot from one gel which is convenient in some cases. To be honest, I also find it simpler than other methods, it takes only half an hour and you can even use the PBS for the blotting.

2- I tried the dot-blot and it worked very well but again, no data about the protein MW.

3- Culture broth: LB-Kanamycin broth with Overnight expression medium  Equilibration/Wash Buffer: 50mM sodium phosphate, 300mM sodium chloride, 10mM imidazole; pH 7.4 Elution Buffer: 50mM sodium phosphate, 300mM sodium chloride, 150mM imidazole; pH 7.4.

4- I don't know that we have different types of column for different starting materials, anyway, I am using this column: https://www.thermofisher.com/order/catalog/product/89971. 

5- Since our protein is tagged with His and MBP tags, we are planning to re-purify it using the amylose resin (against MBP) after the anti-His purification. 

Again, thanks for your joining.

You can also use an anti MBP antibody for detection.

Thanks Vince for your tip,

I think we should , just to reveal this anti-his problem.

Hi Haitham,

Vince was right about an anti-MBP antibody. So what is your control? Is it a free His tag or a MBP-His-protein? Sometimes the position of a his-tag also influence Western.

Also I think your sample is not quite stable with an aggregation band. How did you load your sample? Did you boil it? If you did, try not to by mixing protein and loading buffer then loading it right away.

@ Zhongying,

Thanks for your participation in this discussion,

Yes Vince was right in this point, we have to buy it and see what is goig to happen, my control positive was 15 kDa r-protein tagged with his-tag .

The most right three lanes were of a high protein conc. samples, we use the horizontal electrophoresis , where we load our sample on a filter paper instead of directly in the gell wells, Yes I boiled the samples 95 C for 10 minutes. it is a good tip to follow not to mix the protein with sds before boiling.

Thanks dear Zhongying :)

Could it be that  bacterial lysate contains some  metal binding proteins that non-specifically bind to resin and eluted with the high Imidazole conc.?

It's a possibility, although the metal binding sites of most metal-binding proteins are within the active site and inaccessible to the chelated metal on the resin. A more likely possibility is that proteins containing localized concentrations of His residues on their surfaces bind to the column and are eluted with imidazole.