hi

it seams HRP-conjugated streptavidin is expired or need higher concentrations :

you should ran the positive control in multiple lanes then cut the PVDF and incubated each lane with various dilution of HRP-conjugated streptavidin ( for example 1:5000 to 1:100.000) . in addition you could test the new and fresh conjugate .

"For detection of biotinylated proteins, membranes were blocked in 3% casein in TBS with 0.05% Tween-20 and incubated in the same buffer with HRP-conjugated streptavidin (1:40,000; Invitrogen) "

good luck

Hi Reza Hadavi

Thanks for the input. The Streptavidien-HRP is working becasue the postivie control (biotinylated recombinant protein L) can be detected.

Are those proteins homologs or from the same species? You should check whether addition of the extra biotin increase the endogenous signal. Biotin first should be activated by an enzyme system.

hi

I think this article will help you

Article Screening of Proximal and Interacting Proteins in Rice Proto...

other possibility you need codon optimization according to plant .

good luck

Hi Attila Lehotzky

Thanks for the input. The proteins are from the same species as the protoplast. I'm interested to learn more about the activation of biotin. Could you elaborate a bit on that?

Reza Hadavi

Thanks for pointing out this paper. I am aware of this paper and have designed my positive controls based on their results. I even requested the vectors but had no luck. The BirA used my experiemnt is nucleotide optimized for plant so is more or less similar to their BirAG.

unfortunately, i am not a plant expert. so i not really suggest something to you.

probably i found a link

Article Biotin metabolism in plants

hi

1- if your d-biotin is ok ?

2- use BLRP-GR & BirA co transfect as control

3- tried 16h incubations at 37°C by biotin .

4- If everything was ok and the answers were negative , you should change your construct by Avi-tag, You can order the synthesis of a new vector to solve the problem.

http://www.claudiavickers.org/resources/plant-tools/

Article The pCLEAN Dual Binary Vector System for Agrobacterium-Media...

" Biotinylation of AviTag-fused proteins using BirA

Biotin is very stable so no doubts about that. it will be activated by BirA (intermediate form is biotinyl-AMP) before it is transferred so dont worry about that. what I suspect is that your BLRP-GR fusion protein is not stable and the peptide is removed from the GR by proteases before you can run your sample on SDS page. Can you run samples from cells expressing GR alone and the GR-BLRP fusion in paralell on SDS page to see if there is the expected difference in size? Else, you can try to be very quick in sample pepraration, have everything cold, use protease inhibitors, dentauring agents and all that stuff that may be necessary to prevent proteases to act on your fusion protein.

Dr. Stolz,

Thanks for the comment. I will try to compare GR and BLRP-GR if I have the vectors. I was also suspecting the removal of BLRP. I will for sure add protease inhibitors and PMSF for further analysis.

To elaborate on J. Stolz answer... You mentioned that you detected the GR protein in your extract with anti-GR antibodies correct? In the case of BLRP-GR expression you may be able to see two bands in the western with anti-GR as BLRP adds about 3 kDa to the mass of the protein. You should run untransfected and transfected material and see if you can tell the difference. It would also be wise to include a FLAG or HA tag N-terminal of the BLRP sequence in your BLRP-GR plasmid. This way you can western with anti-FLAG or anti-HA to ensure expression of your transgene.

Second, plants are notorious for containing proteases, that said since you are lysing directly in SDS-PAGE loading buffer (NuPAGE) I don't think the proteolytic degradation is occurring after you make the extract. Doing the experiment described above will ensure not only that you are expressing the transgene but that the BLRP sequence is intact.

Third, use 2% BSA in TBST for your blocking and for applying the streptavidin-HRP to the western blot. I know that you did detect your control biotinylated proteins when using milk but milk does contain some biotin and while it might not be enough to block detection of the biotinylated protein standards it could reduce the sensitivity of the assay such that you are not detected the biotinylation of BLRP-GR.

Fourth, it is absolutely essential to make a fresh dilution of streptavidin-HRP each time you probe a blot. Dilute the Strep-HRP into 2% BSA in TBST immediately before applying to the membrane and use a fresh dilution every time - never re-use. It is not commonly known but HRP loses about 50% of it's activity after even 2 days once diluted in buffer. Often this reduction in activity isn't enough to be noticeable when probing for abundant proteins but it may be enough of a reduction in activity to make detection difficult (i.e. it detects the strongly biotinylated controls but maybe on biotinylated GR).

Finally, you may need to just load more protein. It is also helpful in these situations to run a timecourse if you are able to - determine the time of the maximal expression of your transfected gene product as perhaps its expression has already decreased by the time of your assay.

Good luck. Keep in mind that posting images of westerns and gels will always help your colleagues on ResearchGate to better answer your questions.

Dr. Belak,

I'm pleased to let you know that I switched the blocking reagent to 2% BSA in TBST and it worked like a charm. I detected the BLRP-protein-GR (at the expected size) and self-biotinylated protein from the total protein extract.

Thank you very much for the thorough and comprehensive input. I will write you a private message to exchange contract info so that I could reach out when we publish the work. Thanks again.