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We are glad to carry out Western blotting for you (BP-6). While we have a few antibodies on hand, generally the client supplies the antibody. If the antibody is new and untried, a dot blot test is performed to determine fold dilution. The sensitive ECL method of detection is used (Amersham reagents and protocol). A control membrane run with secondary antibody is included as a control if necessary. If you have already worked out conditions of Western blotting in your lab, order PVDF blotting with blot staining (BP-1 & BP-2). We'll send you the stained, dried membrane so you can do your own Western blotting. We'll help you match up proteins to a duplicate 2D gel if you wish to followup with mass spectrometry fingerprinting. For example, if you want to identify proteins that co-precipitate with the antigen of interest. Figure 1. Coomassie blue-stained 2D gel (~300 ug, left) and PVDF blot (~100 ug protein, right) of Eukaryotic cell line Tera I. Conditions: pH 3.5-10 ampholines, 8% acrylamide slab gels. Presented with permission of Dr Bonnie King, Yale University School of Medicine, New Haven, CT. 2. Can you handle radiolabeled proteins? Yes, we do a considerable amount of 2D electrophoresis of radiolabeled proteins, especially 35S and 32P. We will fax your Radiation Safety Officer a copy of our NRC license on request. If you want to do your own exposures or phosphorimaging, request that the gels be dried onto paper for 35S or 14C. Request Enhance treatment and paper drying for 3H or transfer to PVDF. Staining and transparency drying are fine for 32P if the phosphate bond is stable. Radioisotope concentrations for complex samples should be >25,000 cpm/ul of 35S or 14C for direct autoradiography and 8,000 cpm/ul for fluorography using Enhance (New England Nuclear); the latter assumes the protein concentration is low. Figure 2 shows an example of an 35S-autoradiograph obtained with a 1 uCi load.
Figure 2. Example of 2D autoradiograph. HL-60 cells were labeled for 45 min with 35S-methionine, and the cellular proteins were resolved by 2D electrophoresis at Kendrick Labs. A total of 1 uCi 35S-labeled protein was loaded and the final 2D gel was exposed to Kodak X-ray film for 3 days. Numbers on the left show molecular weight in thousands; the IEF acid end is positioned to the left. Courtesy of Dr. Reba Goodman, Pathology Dept., Columbia University. 3. What is the sensitivity of your Coomassie blue (STP-1), silver stain (STP-2) and Sypro Ruby stain (STP-4)? Our Coomassie blue stain gives a reasonably dark spot at 1 ug and a discernable one at 100 ng. The fluorescent stain Sypro Ruby from Molecular Probes is about equal in sensitivity to our Coomassie stain. For silver staining we use a glutaraldehyde prefix that increases sensitivity and typically gives a reasonable spot at 50 ng and a discernible spot at 5-10 ng. Because of the high sensitivity, the total protein loaded for silver staining is usually kept to 50 ug for complex mixtures for Standard gels and 100 ug for Large Format gels. Even given the reduced load, silver-staining is about ten times more sensitive than Coomassie staining. Figure 3 shows a silver-stained Standard 2D gel loaded with 50 ug of E coli lysate. Note: for mass spectrometry our "special" silver stain (STP-3) must be used; the glutaraldehyde fix is omitted for this stain.
Figure 3. Silver-stained pattern of 50 ug yeast prepared according to our standard protocol with the addition of phosphatase inhibitors. Conditions: pH 4-8 ampholines, 10% acrylamide slab gel with silver stain. The arrow indicates our internal standard loaded at 50 ng. Shown with permission of Dr Irene Ota and Dr Anne Burkholder, University of Colorado, Boulder, CO. 4. What is the maximum amount of protein and volume of sample that can be loaded on your gels? Figure 4 shows the 2D spot pattern of increasing amounts of purified actin. Heavy loading with 5-10 ug of actin does not cause streaking, but rather the spots become larger and more rounded. This only holds true up to a point however; 50 ug of a protein (serum albumin for example) streaks severely and also changes the pH gradient.
Figure 4. Spot pattern obtained when increasing amounts of actin are subjected to 2D electrophoresis with Coomassie blue-staining. Note that the MW line, from the molecular weight standard actin added to the agarose sealing the tube gel to the slab gel runs through the top of the spot.
In general, molecular weight should be measured from a horizontal line drawn through the upper extremes of a spot. In general for complex samples such as cell lysates run on our Standard sized gel, we suggest a maximum load of 200 ug for Coomassie blue or Sypro ruby staining and 50 ug for silver staining. Protein concentrations for complex samples such as cell lysates should be about 5 mg/ml for Coomassie blue and 1 mg/ml for silver staining since the maximum volume that can be loaded is 50 ul. For Large Format gels we suggest a maximum load of 300-800 ug protein for complex samples for Coomassie blue and Sypro ruby staining, and 100 ug for silver staining. The maximum volume that can be loaded is 150 ul. 5. Is there any way to run more than one sample on a gel? Yes, we can run partial samples, (2D-ES-8), where corresponding IEF tube gel sections from different tubes are run on a single 2D slab gel. An example of this is shown in Figure 5. This is a cost effective way to accumulate enough material from a faint protein for amino acid sequencing.
Figure 5. Four corresponding tube gel sections were run on one slab gel for purposes of comparison. 6. How do you
standardize pI and MW? In part our service is possible because 2D gels may be conveniently
standardized. We
used to (and sometimes still do) add molecular weight (MW) markers to the
agarose sealing the tube gel to the slab gel, to make a final MW grid. While
this worked nicely for manual comparisons, with the advent of computerized
comparisons and mass spectrometry, the MW markers are best run down the edge of
the pattern as shown in Figure 6 below. A single isoelectric focusing
standard,
tropomyosin of
pI 5.2 and MW 33,000, is added to every sample as a control and is marked by
an arrow on the gel. Figure 6. Mouse
heart homogenate. The molecular weight standards run in a lane on the right
edge of the gel are myosin, 220,000; phosphorylase A, 94,000; catalase, 60,000;
actin, 43,000; carbonic anhydrase, 29,000 and lysozyme, 14,000.
The pI standard marked
by an arrow is tropomyosin, a doublet with marked
spot of pI 5.2, MW 33,000. This gel
has been silver stained. 7. How are isoelectric points determined? A pH gradient plot obtained with a surface pH electrode and six IEF tube gels (run with buffer only) is returned with every set of gels to aid in determining pI's of specific proteins. The exact size of a gel varies with treatment: Enhanced gels are larger, silver-stained gels a bit smaller than Coomassie blue-stained gels. To determine the approximate pI of your protein of interest, adust the size of the pH gradient plot on a photocopier to fit your gel, overlay the plot on the gel on a light box and read the protein's pI off the plot. In some cases, salts and buffers in samples may enter the focusing gel and change the pH gradient. When necessary, the carbamylated protein sets of carbonic anhydrase (CA) or creatine phosphokinase (CPK) shown in Figure 7 and calibrated by Kendrick Labs may be added to samples to exactly determine a protein pI. Radiolabeled carbamylated CA is also available. Note that the isoelectric point of a protein is not a constant value like molecular weight but is dependent on conditions such as pH, buffers and temperature. Our pH gradient plots are for conditions of 9M urea and 22o C.
Figure 7. Example of carbamylated CA and CPK pI markers. The arrow indicates our internal standard tropomyosin added to every sample unless otherwise requested; numbers to the left show MW in thousands. 8. How do you identify proteins of interest on 2D gels; how much material does it take? Protein identification is typically achieved by either mass spectrometry fingerprinting (e.g. Cottrell, S and Sutton, C, "Protein and Peptide Analysis by Mass Spectrometry" pp 67-82, In: Methods in Molecular Biology, Vol 61, 1996, ed. John Chapman) or amino acid sequencing by LC/MS/MS (Protein Sequencing and Identification Using Tandem Mass Spectrometry, Michael Kinter and Nicholas E. Sherman, Wiley, John & Sons Inc, 2000). Any polypeptide spot, however faint, on a Coomassie blue-stained gel is in range for identification by mass spectrometry. Reasonably dark prokaryotic proteins on a "special" silver-stained gel (STP-3 in Guide to Services) are also within range for mass spec. One problem with amino acid sequencing by LC/MS/MS is that the sequences tend to be only 5-10 amino acids in length for several peptides. If longer sequences are needed for creation of a probe, then sequencing by Edman degradation is still useful as it usually gives 12-20 AA sequences. At least 10 times more material is required for amino acid sequencing by Edman degradation (e.g. Matsudaira, P "Chapter 1, Overview" pp1-13, In: A Practical Guide to Protein and Peptide Purification for Microsequencing, Ed. Matsudaira, P, 1993.) than mass spectrometry. The rule of thumb is that a polypeptide spot must be fairly dark with Coomassie blue-staining on a 2D gel to be in range for sequencing. The tropomyosin standard spot (arrow in Fig 3) is an example of the lower limit of material required for amino acid sequencing. It may be necessary to combine several corresponding spots from separate gels to get enough material. N-terminal sequencing is done from Coomassie blue-stained PVDF blots while internal sequencing is done from Coomassie blue-stained spots cut from dried 2D gels. Often amino acid sequencing identifies proteins that are unidentifiable by mass spectrometry fingerprinting through homology. For example a protein of interest from Elk serum could not be identified by mass spec fingerprinting since few Elk proteins are in Genbank. However, an eleven AA sequence from the protein showed clear homology with human and mouse complement factor B. Even if a protein cannot be identified, a peptide sequence, usually 10-20 amino acids, can be used to generate a probe for finding the gene. We don't carry out mass spectrometry or amino acid sequencing in house but have considerable experience in submitting samples to university core facilities. We are happy to handle all aspects of submission of your protein spots including spot cutout, express mailing to the core facility and invoicing. Our default core is Columbia University Protein Core Facility in New York ( http://cpmcnet.columbia.edu/dept/protein )Spot cutout is based on your emailed image, or on the dried gel overlaid with a transparency on which the spots of interest are marked. After spot cutout, the dried gels are returned to clients along with a copy of the letter sent to the facility. Turnaround for results is usually 2-3 weeks. Home Return to TopFeel free to call or email to discuss your project or for a price quote at no obligation.
Telephone:
Email: 2d@kendricklabs.com
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