Antibody Issues - (Feb/11/2015 )

Hi- wondering if someone out there can help figure out what's going on with my WBs!

So I have been transfecting one set of HuH-7 hepatocarcinoma cells with a plasmid of interest, and another set of HuH-7 cells with a negative control plasmid. These cells were lysed at 48 hours and at 72 hours.

 

I have been using Santa Cruz antibodies to seek out Protein S (72kDa), TFPI (40kDa), C4BP-a (70kDa) and C4BP-b (52kDa). Protein S is Mouse anti human, TFPI and C4BP-b are Goat anti human, and C4BP-a is Rabbit anti human. The proteins antibodies had to be optimised, and it was determined that TFPI is running higher than expected at about 50kDa. Actin was used as the housekeeping protein.

 

I have been using SDS-PAGE to transfer the proteins to a nitrocellulose membrane overnight (approx. 17 hours at 4 degrees Celsius). Then I checked each membrane with Poinceau Red staining, before applying a 3% Milk/TBS blotto for 90 minutes. The 3% is removed, and the Primary antibody (dissolved at the required concentration) in 1% Milk/TBST blotto is poured on for 90 minutes. The next step is to do 3x 5 minute washes of TBST, before pouring on the Secondary antibody/HRP and Streptavidin (again dissolved at the required concentration) in 1% Milk/TBST blotto. The Secondary antibody is left to wash the membrane for 90 minutes; it is then poured off, and the membranes undergo another set of 3x 5 minute washes. The membranes are blotted dry, and ECL fluid (from Bio-Rad) is pipetted onto them. The ECL fluid absorbs light for 5 minutes, and then the membranes are blotted again to remove the excess fluid before being sandwiched between two clean and clear plastic sheets. Finally, they get to be imaged at 30 second intervals for 2 and a half minutes.

 

During the optimisation process, the antibodies worked really well for Protein S, C4BP-a and TFPI. But then when I used the cell lysates, I end up with a non-specific band around 100kDa, across all antibody types. It's a very strong band. Protein S and C4BP-a both appear, however there are non-specific bands below them at about 60kDa. The TFPI and C4BP-b columns don't produce any bands.

 

The results are consistent across all the membranes I've trialled, however they aren't consistent with previous studies or with my optimisation blots. Sometimes the Molecular weight markers disappear entirely.

 

My supervisor and I are confused as to why a non-specific protein is binding to antibodies from 3 different species, and as to why the C4BP-b and TFPI don't show up, considering they are both linked to Protein S which is showing up very clearly.

 

Thanks in advance if you can think of anything, and if you seek clarification on anything, please ask!

 

(PS- I am still awaiting permission to upload the WB images)

non-specific bands around 60 kDa are most likely keratins from dust.

 

if you are seeing the 100 kDa band for all antibodies then it is probably binding the secondary antibody (or the streptavidin, why do you add streptavidin if your secondary has hrp already bound?).

 

you may be able to eliminate that band by adjusting the secondary antibody concentration.

 

the primary antibodies that don't show any banding for the proteins of interest may need to be optimized or replaced.

Hmm, the environment is not very dusty, but I have been using ethanol to spray the plastic sheets. I wait for it to completely evaporate first before I place the membranes on, so it shouldn't have any effect on the membrane?

From what I understand, the secondary antibodies/HRP alone doesn't show up in our imager. The Streptavidin is required to further enhance the protein antibodies as well as the ECL fluid. It may be that the Streptavidin is binding to the non-specific proteins, and as such is being imaged at 100kDa.

 

We've ordered new antibodies from a different company, so we'll see how that goes.

I will raise the theories about dust and increasing/decreasing the secondary antibody- I still find it weird that ALL the antibodies are binding to the non-specific protein when they're all from different origins and should be specific to the proteins of interest. Hopefully the new antibodies work, and I will try lowering the concentration of Streptavidin to see if that has any effect.

Thank you for the reply!

Keratins from dust are present in anything that could be exposed to dust/skin particles and are a very very common protein to find in western blots, so it is nothing to do with spraying the plastic trays. The keratin is actually in the gel from you making the gel and also from loading buffer and cell lysate preparation etc, and then gets transferred to the membrane (hence it comes out as a band).

 

Streptavidin only increases specificity of the interaction if biotin is present in the protein of interest, the strept/biotin interaction is highly specific and very strong. If you lack either one of these parts then it will do nothing for increasing specificity of the interaction, but is more likely to cause background staining.

 

There is a chance that something has contaminated your antibody stocks (bacteria?) that could be causing a non-specific interaction to your 100 kDa band.

Thanks bob1- the gels I've been using are preset, so I'll have to run a dummy gel to see if there are any random bands around the 60kDa and 100kDa marks. It is possible that if keratins are present that they entered the cell lysates when they've been sitting on the lab bench.
 

Will discuss with my supervisor about the need for streptavidin.

 

There shouldn't be an issue with bacteria as the lab is PC2 certified- if there are bacteria present in the fridge then we could have a big problem. For the new blots I have been using a new batch of Western Loading Dye, so there may be a contamination issue there.
 

UPDATE: I'm in the process of testing for the housekeeping protein (Actin), and have noticed something interesting. When I removed the membranes from the fridge, the molecular weight markers had turned brown rather than their usual blue and pink. The non-specific banding at 100kDa was also brown. Can't really find any  information as to why the MWM has changed colour, nor why the non-specific band is still present when it should have faded overnight.

Tayzz on Thu Feb 12 02:29:18 2015 said:

Thanks bob1- the gels I've been using are preset, so I'll have to run a dummy gel to see if there are any random bands around the 60kDa and 100kDa marks. It is possible that if keratins are present that they entered the cell lysates when they've been sitting on the lab bench.
 

Will discuss with my supervisor about the need for streptavidin.

 

There shouldn't be an issue with bacteria as the lab is PC2 certified- if there are bacteria present in the fridge then we could have a big problem. For the new blots I have been using a new batch of Western Loading Dye, so there may be a contamination issue there.
 

as bob1 said, there is dust everywhere. in one of the papers which identified the contamination, the author introduced the bands by briefly sticking their finger into the electrode buffer.

 

are your secondaries biotinylated and you add streptavidin and biotinylated hrp? that would be why you can see results with streptavidin and don't without.

 

you must determine if your secondaries are binding to the 100 kDa band rather than your primaries. you should also determine if, although unlikely, the 100 kDa band is directly reacting with the ecl reagents.

 

Tayzz on Thu Feb 12 02:30:25 2015 said:

UPDATE: I'm in the process of testing for the housekeeping protein (Actin), and have noticed something interesting. When I removed the membranes from the fridge, the molecular weight markers had turned brown rather than their usual blue and pink. The non-specific banding at 100kDa was also brown. Can't really find any  information as to why the MWM has changed colour, nor why the non-specific band is still present when it should have faded overnight.

the color changes are probably due to reaction with the ecl reagents.