Hi Kaija,

normally there is no need for blocking, neither BSA or Casein. Strept(avidin) coated plates (normal concentration is between 2 - 10 µg/mL) in a 0.1 mol/L carbonate buffer pH 9.6, overnight incubation at 4 °C, removal of the coating solution (as much as possible) can be stored frozen without any problems. Before using them for the second coating step with the biotinylated peptide you should use already a Tween 20 containing buffer (PBS). The detergent is responsible for blocking. Only if long time storage at room temperature is required, BSA + sugar (mannitol or glucose 0.1% w/v can be helpful).

If you have problems with free biotin-binding sites, there are three opportunities: 1. increase the concentration of the biotinylated peptide, 2. use free biotin or us a simple biotinylated amino acid like glycin.

Incubation steps with antibodies, serum dilution etc. need the protein content (at 1 - 3% w/v) to keep the protein concentration under different dilutions constant. Otherwise you are on risk to detect simply matrix effects due to the dilution. Therefore BSA is a good choise, from a hospital you can take protein / colloid containing drugs like human albumin, gelatin (hydrolysed), hydroxyethly starch. Those buffers should contain also Tween 20.

We are preparing a PBS, 0.1% Tween 20. This buffer can be used as washing buffer. Add some BSA, HSA etc. and you will have your incubation buffer. So you can reduce the amount of protein to a minimum. Just a last trick: If you add some phenol red (1 mg/L), you will see much better where you are during pipetting, this solution is colored.

My only thing to add is to look for a pure BSA to be sure there is no biotin in it. Be careful with using any serums, which will contain biotin. Another option is using using a gelatin mixture; 1-5%.

Hi Kaija,

normally there is no need for blocking, neither BSA or Casein. Strept(avidin) coated plates (normal concentration is between 2 - 10 µg/mL) in a 0.1 mol/L carbonate buffer pH 9.6, overnight incubation at 4 °C, removal of the coating solution (as much as possible) can be stored frozen without any problems. Before using them for the second coating step with the biotinylated peptide you should use already a Tween 20 containing buffer (PBS). The detergent is responsible for blocking. Only if long time storage at room temperature is required, BSA + sugar (mannitol or glucose 0.1% w/v can be helpful).

If you have problems with free biotin-binding sites, there are three opportunities: 1. increase the concentration of the biotinylated peptide, 2. use free biotin or us a simple biotinylated amino acid like glycin.

Incubation steps with antibodies, serum dilution etc. need the protein content (at 1 - 3% w/v) to keep the protein concentration under different dilutions constant. Otherwise you are on risk to detect simply matrix effects due to the dilution. Therefore BSA is a good choise, from a hospital you can take protein / colloid containing drugs like human albumin, gelatin (hydrolysed), hydroxyethly starch. Those buffers should contain also Tween 20.

We are preparing a PBS, 0.1% Tween 20. This buffer can be used as washing buffer. Add some BSA, HSA etc. and you will have your incubation buffer. So you can reduce the amount of protein to a minimum. Just a last trick: If you add some phenol red (1 mg/L), you will see much better where you are during pipetting, this solution is colored.

be careful with serum in your incubation buffers. It's difficult to keep the quality of the buffers constant. There are sometimes sera from particular animals who did not work. In some cases, also antibodies against strepavitin are present. So check it carefully with the respective negative controls.

Hi Kaija,

PSBT (PBS with 0.1% (w/v) Tween 20) for blocking is fine (and usually sufficient). Simply add 250µl/well and set aside for 15mins. If you should get a background later, consider diluting antibodies further. As standard BSA may contain pretty much biotin, there is some risk that the relatively huge BSA molecules attach to the streptavidin layer and mask your biotinylated peptides by rendering them unreachable for their intended targets. If concerned about unbound biotin binding sites, simply block them with a slightly alkaline solution of biotin in your preferred buffer.

BTW: would you like to share your protocol for coating the plates with streptavidin?

Thanks & HTH! Wolfgang

We keep it very simple and substitute non fat dry milk (2%) for the casein and have not experienced any difficulties with biotin contamination.

It's very easy, Kaija! I recommend you use PBS with Tween 20, and later, in the same moment, you can add dry milk 2%. I always do it so, very luck for you!

I have always used BSA (PBS, Tween 20 + BSA) as a blocking agent and is fine. I agree with Dr. Ezio Petrella about BSA purity and with Dr Walter Papasergi in trying different concentrations of BSA. I suggest start with 5% and always use a negative control.

Good luck!

You can try to use the milk (non-fat) it is the think more close to the casein. Anyway I would not change the protocol suggested from the plates producer, you risk to waste your time in set up new condictions.

Just to know, in my ELISA based on E.coli expressed proteins I use 2h blocking in 3% milk-PBS and for human/mouse serum dilution 1% milk-PBS .

Ciao!

Thanks so much everyone!!

As the streptavidin plates were bought pre-coated, their manuals suggest they don't require additional blocking with caseinate or BSA, just the PBS-Tween20 wash before addition of peptides.

Walter- As the last plate I ran didn't seem to have abnormally high background, we've decided to stay with the simple wash before use. The only issue we had was that the human serum results were higher than maximum absorbance.

Wolfgang- the peptide manufacturer gave the attached instructions for coating the plate, and had we not ordered the plates, we would have performed it this way.

http://www.mimotopes.com/files/editor_upload/File/PeptidesAndAntibodies/PU3005-1Assays-of-Biotinylated-Peptides.PDF

You have several ideas and tips now, but my advice is to ry and see. Yes use a pure BSA or sodium caseinate and compare the results for precision, accuracy, cross-reactivity and sensitivity. I use about 1% BSA for most of my steroid ELISAs.

Hi Kaija, many thanks for the instruction sheet! It is very helpful!

Yes if you havn't caseinate can use BSA but only V fraction.

@Emad

How are you dealing with the background caused by steroids from the donor animals?

KISS: keep is simple and stupid.

It doesn't matter which protein you take.

Something about the background:

1. In most cases it's not necessary to block the plates. Blocking is done during the first washing with PBS Tween (by the detergent). The hydrophobic ends of this molecules will bind to the hydrophobic styrol groups at the plate.

2. Antigen coated plates can be stored frozen until use.

3. Only for commercial reasons (or if long time storage is required, under dry conditions in sealed bags) blocking is required. In this case add some sugar (1% mannose + 1% glucose) can be helpful.

If you are using detergents for long time storage, this molecules will replace slowly (but surely) the proteins from the binding at the solid phase. Therefore you will lose some sensitivity.

Stephan

Thank you Stephan- that was very helpful and puts my mind at a little more ease!

BSA (1-3 %) should normally work well here. I rather would be reluctant using milk dreived proteins like casein on streptaviding plates, as these might more likely contain biotinylated protein contamintations than BSA does. Bodo

There are some difference for different blocking agents (BSA, casein, non-fat milk, tween 20, sucrose etc)  in my ELISA array based experiment. Casein give the best S/N ratio.

I recently encountered a protocol that used HSA and FSG as a blocking system. I found it a bit unusual to be using both as I would assume FSG to be quite effective on its own. I'd also suspect HSA to be susceptible to interference. Has anyone here ever used this combination or does anyone have any useful tips?

Thanks,

Alan