::Affinity Photoprobes, LLC::

Question #1:
Do I have to handle photoprobes in a dark room or under reduced light?

Answer:
No. Nucleotide photoaffinity probes are not activated by normal room lighting (fluorescent or incandescent).  However, avoid exposure to UV light or sunlight prior to use.

Question # 2:
What type of UV source is ideal for these irradiating nucleotide photoprobes?

Answer:
A hand-held UV lamp (254 nm) with an intensity in the range of 2000-6000 µW/cm² is ideal for photolyzing azido photoaffinity analogs.  UV crosslinker ovens will also work.  However, the photolysis time required for optimum photoincorporation should be determined.

Question #3:
Why are all the photoprobes shipped as solutions in absolute methanol and how do I get the photoprobe in the aqueous buffer that I want?

Answer:
Azido nucleotides are most stable when stored as a solution in methanol in -20^oC and should be stored at this temperature upon receipt.  To resuspend the photoprobe in the experimental buffer, pipette the aliquot required in a micro-tube and remove the methanol in a vacuum concentrator or under a gentle stream of nitrogen gas in a fume hood.  When dry, an appropriate volume of buffer is added and the photoprobe resuspend by mixing with a vortex.  Never add methanol directly to your purified proteins, tissue homogenates, cells or cell lysates.  Methanol precipitates protein.  Take 5 minutes to dry your photoprobe and remove the methanol. It will be the best 5 minutes you ever spend in lab.

Question #4:
Will I observe photoincorporation in the presence of reducing agents such as DTT and mercaptoethanol?

Answer:
Azido nucleotides are reduced to non-photoactive amino nucleotides in the presence of reducing agents.  Therefore, a reduced photoincorporation will be observed in the presence of DTT or b-mercaptoethanol.  If a reducing agent is required to maintain protein activity, cysteine at concentrations less than 100 µM can be used as long as the photoprobe is not incubated for extended periods of time in the presence of the reducing agent prior to photolysis.

Question #5:
I remember from past literature that the photoinserted bond formed between P-32 labeled nucleotides and proteins and peptides is sometimes not stable to reverse phase HPLC conditions.  Are there any technical advances in the area of isolating active site peptides that help preserve the radiolabel?

Answer:
Yes. Reduced flow rates (0.5 ml/min compared to 1.0 ml/min) during RP-HPLC purification helps to retain close to 80% more label with the photolabeled peptide.  This particular observation along with the use of immobilized metal affinity chromatography (see IMAC) has helped in the isolation of active site peptides. 

Question #6:
When working with crude samples (tissue homogenates, cell lysates, biological fluids, etc.), how do I photolabel my low abundance nucleotide binding protein of interest in the presence of other more abundant nucleotide binding proteins?

Answer:
Many nucleotide binding structural proteins such as tubulin (GTP binding) and actin (ATP binding) together with house keeping proteins such as pyruvate kinase, phosphoglycerate kinase, creatine kinase, etc. are present in very high amounts relative to low abundance proteins of interest such as G-proteins and certain regulatory kinases.  In these cases, it is best to purify or concentrate the protein of interest.  One of the easiest methods of achieving this aim is by subcellular fractionation of the crude preparation.  Another option is to concentrate the protein of interest by ammonium sulfate or polyethyleneglycol (PEG) precipitation.

Question #7:
What are the three most important things to remember when performing any nucleotide photoaffinity labeling experiment?

Answer:
Optimize, optimize, optimize!!  Don't assume that the experimental conditions that you have been using to perform your enzyme assays are the optimal conditions for photolabeling.  Photolabeling, in contrast to enzymatic turnover, measures only the initial binding of the nucleotide to the protein.  This protein-nucleotide interaction can be dramatically affected by your choice of buffer, pH, ionic strength, presence of metal ions, cofactors, photoprobe concentration, time of incubation of enzyme with photoprobe, etc.  Take the time to optimize these variables.  In general, a good place to start is with 10-20 mM Tris-HCl or sodium phosphate, pH 7.0-7.5. with 1.0 mM MgCl₂.  A 30 sec incubation of the photoprobe with the protein followed by a 60 sec photolysis is usually sufficient but these two variables should also be optimized to achieve maximum photolabeling efficiency.

Technical Information or General Inquiries

Contact Dr. Anjan Bhattacharyya, Ph.D.
Radiochemicals Laboratory Director
E-mail: ajbhatta@Photoprobe.com