Hi
can anyone help me ???
I did 4 rounds of biopanning with my antigen. I got 2 times more colonies in test ( where antigen was coated ) as compared to blank ( no antigen ). after that i performed Phage ELISA but i am not getting any difference with or without antigen in the development of color after adding substrate. when i compared the absorbance then there was minor difference ( 0.060 to 0.100) in the OD values. i have used alternative blocking agents during biopanning and different blocking agent in Phage ELISA. Tween 20 concentration was 0.1 % and did 10 times washing during biopanning steps. what could be the problem ??
Your panning failed. After 4 rounds of panning, you should see >10x binding on antigen versus blank. You probably need more stringent panning, because phage are very sticky. Try PBST containing 1-2 mg/mL blocker protein (or up to 1% serum), plus 0.1 M glucose. If your antigen is highly charged try increasing NaCl to 250 mM, if it's hydrophobic try increasing Tween to 0.25%. After loading your pan with antigen, and rinsing, be sure to preblock for a few seconds before adding phage diluted in blocker.
@John Carter
I Have coated antigen for overnight at 4 degree and then did washings with PBST (tween 20 =0.1%). After that I did blocking for two hrs with 3% skim milk at room temperature and then added phage for binding.
If i understood your suggestion correctly then you means I should use blocker protein and glucose in PBST for washing!!
Your antigen coating step sounds good.
Apparently milk protein (casein) is not working for you. So try 1-3% BSA or ovalbumin, or 1-10% serum. Glucose sometimes helps a little.
I agree your panning probably failed. Just to make sure, do you have any positive control to confirm that the phage supernatants you used for phage ELISA actually contain phages? If you test them on plates coated with an anti-M13 mAb, you can rule out a failure in phage production at 96 well scale (I guess this is what you did before the phage ELISA). If the molecules you are trying to select from the library (for instance antibody framents) are fused in your constructs to a tag (for instance c-myc peptide), then you can also test the phages on plates coated with an anti-tag mAb. A positive result would tell you not only that the phages were produced, but also that the foreign proteins were displayed. Such controls could help you to be sure that the problem is not related to the screening ELISA and your clones are really negative.
About the panning, I always use 4% skim milk to block the tubes coated with the antigen, but I also pre-block the phages with the same blocking solution. Then I add the phages (diluted in PBS/milk) to the coated tubes. It reduces the panning background. I always wash with PBS/0.1% Tween 20 without further additives.
Although most protein antigens are very easily used to coat inmunotubes or other surfaces, there are exceptions. Did you check by ELISA with specific antibodies that your antigen is properly coating the surfaces used for phage panning and screening? This is really critical.
Titration results are often useless to predict panning success. And a difference of 2-fold (antigen-coated vs non-coated) is to low to be taken into account. So, focus on the ELISA results and forget the titrations.
@Gertrudis Rojas
Phage particles are being produced because i am using anti pVIII M13 antibody which is giving positive color with test wells. As such this antibody ( anti pVIII ) doesnot bind to empty wells. so i guess this ensures that phage particles are being produced. Next thing is tagged insert . here problem is that my vector doesnt contain any tag . After reading your suggestions i guess i should check the display part.. If i coat 96 well plate with anti pVIII antibody and then put Phage , after that use pIII antibody but again it wont tell me whether display of fused protein is happening or not ... may be i should do western blotting for phage particles displaying fused protein ( for atleast few clones), change in band molecular weight will may help!
For antigen stability , i have checked that it is not degrading during ELISA steps .
I didn't understand the test you are doing wih the atni-PVIII.
Anyway, yes, you can check display by WB, although it would be easier to do it by ELISA (having a tag). Are you producing phages in the absence of glucose (in case you are using pLAc or a similar system)? Otherwise glucose can downmodulate the expression of the foreign protein.
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