First question is: are you sure the 3F4 epitope was conserved in your PrPC species? Usually the 3F4 is a great anti-PrPC antibody, perhaps one of the most reliable and efficient...

Look at the paper of Oelschlegel A et al or Gretzschel A et al. http://www.ncbi.nlm.nih.gov/pubmed/17098989  or check for Groschup MH. there are a couple of paper's from that group that stained the prion protein with 3F4 antibody, both PrPC and Sc.

Dear Emiliano 

I am working with human cell line which is U87 for GBM.  

Did you try to compare with another antibody? (like 6D11, 8H4 or D18) May be PrPC is poorly expressed in your particular cells...

I have got a result with 8H4 but this antibody exhibited a bad background. Also 8H4 gave a clear band with another protein in approximately 80 KD.   

Try 6H4 we found it works well in our hands, coupled with a HRP secondary, enhanced chemi kit and developed on low light camera

what gels, secondary, and detection substrate do you use?

Element of discussion:

The PrP antigen sequence acting as an epitope of the 3F4

Furthermore, information was acquired from antibody

investigations aimed at blocking the transformation of PrPc

into PrPsc. The PrP antigen sequence acting as an epitope

of the 3F4 antibody, varies from 4 to12 amino acids,

depending on the animal species (Lund, Olsen, Tveit, &

Tranulis, 2007; Zou et al., 2010a), 3F4, a monoclonal antibody

(MAb), has been recognized as a reagent that greatly

facilitates research on PrP and prion diseases (Zou et al.,

2010a). The minimum sequence consists of the

109MKHM112 tetrapeptide common to human and hamster

PrP and the same sequence with different numbering

occurs in other species (Zou et al., 2010a). The same case

is applicable to the heptapeptide 106KTNMKHV115 for

bovine and elk PrP (Zou et al., 2010a) and

106KTNMKHM115 (Lund et al., 2007) for humans and

hamsters and 104KPKTNMKHMA113 with an Ω loop

structure. (Determination by X-ray (Kanyo, 1999).

These amino acid sequences are not sterically accessible in PrPc, but can be presented to 3F4 in PrPsc (Figure 7). The Lys106 residue is proved to be a critical residue as shown by the marked reduction of 3F4 binding observed after a deletion in the 12-mer PrP peptide 107 (Lund et al., 2007; Zou et al., 2010a). However,

3F4 remains the most important monoclonal antibody in

studies related to human prion disease, and it confirms that our miniprion_beta PrPsc 92–138 can easily enter the 3F4 epitope, allowing immunoreactivity with the minimum sequence 106KTNMK110 (humans) and 106KTNLK110 (mouse) (Lund et al., 2007). The epitope shown in Figure 7 is no longer accessible in nondenatured PrPsc, showing that PrP transconforms during

scrapie infection and adapts to epitope 3F4. A large

portion of the PrP molecules was subjected to proteolytic

processing, giving a positive reaction with 3F4 (Pan

et al., 2002). Recently, a letter of reply to Kascak (2010)

answered by Zou et al., (2010a) gave general agreement

that 106KTNMK110 lacks Met112 (Zou et al., 2010b),

suggesting that these five residues constitute a minimal

3F4 epitope. Figure 7 further on shows the trace of the

putative epitope in our model.

 (See figure 7 in: Yves Chapron*, Laurent Charlet and Nita Sahai. 2014, Fate of pathological prion  (PrPsc92–138) in soil and water: prion-clay nanoparticle molecular Dynamics.  Journal of Biomolecular Structure and Dynamics, Vol. 32, No. 11, 1802–1816)

Hi Ibrahem,

Als the otters already mentioned 3F4 is a great antibody.

But you didn't mentioned on what species you workshop, for 3F4 works best on Human and Hamster PrP(Sc).

Second, I don't know what technique you're using to detect it and what kind of experiment you did (i.e. digestion with pk)..

We have more information available about which antibody binds where on the PrP molecule: see out website:

www.wageningenur.nl/prionantibody

(about our prions-related PrP- specific antibodies 12B2, 3A2, 9A2, 6C2, 100B3 or 94B4)

I have attached a pdf file with all the different antibodies and their binding-epitopes als wel.

We would be happy to help you further.

Jan Priem

Dept. Bacteriology & TSE's

Centraal Veterinary Institute of Wageningen UR