Peptide Array Serives

Providing the most cost effective peptide synthesis and innovative protein array technology

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Frequently Asked Questions
	1. Question: For probing how much purified protein or recombinant protein do I need to supply you with to screen the peptide library? Answer: Depending on your request you will need to provide us with 6-180 ug of purified or recombinant protein. If you use a small molecules ie. peptides you will need about 2 mg of the purified substance.
	2. Question: For probing, does my protein need to be labeled or can I just supply you with an antibody to my protein? Answer: You may send us a labeled protein ie. biotinylated or an antibody to your protein for detection purposes. If you have a cell line with your protein of interest then we would need an antibody to detect the protein in the mixture (quantity of antibody required: 1:1 ratio amount to the protein probe)
	3. Question: How many binding partners do you usually detect per screen on the arrays? Answer: Depending on the screening type we detect between 5-20 peptide binding partners
	4. Question: How do I test the peptides once I have identified possible binding partners? Answer: It all depends on your experiment, most do western blots with the corresponding labeled peptide or functional assays.
	5. Question: How are the probing results presented to me? Answer: We will provide you with the sequences of the identified binding partners then you may have us synthesize them if you wish to test them.
	6. Question: How are the data from probing evaluated? Answer: Detection is by enzymatic development of a color, chemiluminescence or fluorescence using any of a variety of methods similar to those used in developing western blots. In addition, radiolabeled probes may be used for example to directly determine sites of phosphorylation using a kinase and ATP32. Arrays may be scanned using a flatbed scanner or by exposure to Xray film. A typical screen of peptides would involve probing for example with a mouse antibody whose epitopes one wishes to determine followed by a labeled second antibody such as HRP conjugated rabbit anti mouse antibody.
	7. Question: How is the quality of the array synthesis monitored ? Answer: There is no validation of the quality of peptides during each run. We have established peptide quality on several previous occasions by HPLC and Mass Spec. identification. In general, the quality has been greater than 70% for peptide purity.
	8. Question: How long should my peptides be? Answer: Peptides can up to 20 mer in length however we found that 12mers are the ideal length.
	9. Question: What is shifting? Answer: Shifting means that if peptide # 1 contains sequence aa 1-12 of a protein then if we shift by 2 amino acids the second peptide will have sequence aa 3-14. This is to ensure that we isolate the exact amino acid sequence that the probe is binding to. We find that shifting by 2or 3 is ideal. Ex. HRFACGQPMNSI FACGQPMNSICG CGQPMNSICGLW If three consecutive spots are positive for example for the above three peptides, then the epitope is assumed to be CGQPMNSI which is common.
	10. Question: How many copies of arrays do I need to make? Answer: You should make two copies, one copy for your control (ie. Probe with just your antibody) and one copy for your sample (ie. Probe with protein and antibody)
	11. Question: How do I calculate the number of peptides in a Pep-Array? Answer: The number of peptides depends on the number of aa in the protein, the peptide length and the shift. To calculate the number of peptides in a Pep-Array use the following formula: [(#aa in the protein – peptide length)/(shift)] + 1 = #peptides
	12. Question: How do I calculate the cost? Answer: The cost depends on the peptide length and the shift use the following formula to calculate the number of peptides: (#peptides) x (peptide length) = total # aa (Total #aa) x (cost per amino acid) = cost
	13. Question: How are the results from the arrays calculated? Answer: The results are calculated by determining the signal intensity of the probed peptides. The control experiment (The intensity of the detection agent without the target protein) is subtracted from the sample intensity in order to ensure that the binding is strictly through the probe. The results will be given to you in terms of intensity values.
	14. Question: How do you eliminate unspecific binding? Answer: We block unspecific binding with 5% skim milk in T-PBS or T-TBS buffer.
	15. Question: How are the peptides synthesized? Answer: Synthesis is carried out using standard Fmoc chemistry. Peptides are assembled amino acid by amino acid.The α-amino Fmoc protection group ensures that at each step only a single amino acid is coupled. Some amino acids require side chain protection. The Fmoc amino acids are purchased with side chain protection where necessary. The Fmoc group is removed after each cycle ie. following coupling of one amino acid thereby exposing a free amino group. The next Fmoc protected amino acid in the sequence is coupled via its carboxy terminus again. After the last coupling cycle when the entire peptide has been assembled, the side chain protection groups are removed.
	16. Question: At what temperature should the peptides be stored at? Answer: We recommend that you store the peptides at –20 ° C.
	17. Question: C-terminal amidation? Answer: This is to avoid unnatural charges that may be found in the native protein as well as to avoid the peptide to cyclize on itself.
	18. Question: I am having problems with the solubility of my peptides, what can I do? Answer: Depending on the amino acid sequence your peptide will vary in solubility it often helps when dissolving it in your buffer of choice to vortex or sonicate the peptides. For hydrophobic peptides try dissolving the peptide in an organic solvent such as DMSO.
	19. Question: How will the peptides survive in vivo? Answer: Cyclyzation or using D amino acids will make the peptide more stable and resistant to degradation.