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Carol Browner, Administrator
Public Information and Records Integrity Branch (PIRIB)
Information Resources and Services Division (7502C)
Environmental Protection Agency
1200 Pennsylvania Ave NW
Washington, DC 20460
RE: DOCKET # PF-867B
Dear Ms. Browner,
I am writing on begalf of the Biotechnology Industry Organization (BIO) in support of the Supplemental Safety Assessment submittedby Aventis. The data in the Supplemental Safety Assessment reinforces
the conclusion that the Cry9C protein in StarLink corn is not an
allergen and is not likely to lead to an allergic reaction. The proteins
that cause an allergic reaction have certain physicochemical
characteristics in common. The Cry9C protein does not have these
characteristics.
The most important characteristics are those which effect the quantity
of protein likely to be present in the gastro-intestinal tract. Both
stability to digestion and level of protein together affect this
quantity. Significantly, the Cry9C protein represents an extremely
small amount, only 0.0129 percent of the total protein in grain from
StarLink corn. In contrast, typical food allergens on average represent
between 1 percent and 40 percent of the total protein in the raw food or
grain.
It is also worth noting that in a rodent toxicology study, no adverse
immunological effects were observed at the very highest dose tested,
which implies that Cry9C is not easily or adversely perceived by the
immune system. Furthermore, presently corn-allergic individuals were
shown by Aventis to be no more sensitive to StarLink corn than to
traditional corn, which leads to the conclusion that StarLink corn is
not a threat to these "at risk individuals." Protecting food allergy
patients from unwanted and unexpected sources of allergens which cause
their illness represents an important public health dimension to any
allergy assessment of foods, whether traditional or derived through
biotechnology.
Aventis has requested a time limited exemption from the requirement of a
tolerance for the Cry9C protein expressed in StarLink Corn in food. This
clearance would cover any StarLink corn grown in 1999 and 2000. Also, since
Aventis has withdrawn its EPA license for StarLink, there will be no further
production of this corn in 2001. Therefore, there is a completely new context for
granting a temporary exemption from the requirement of a tolerance for
Cry9C protein in StarLink corn, which is that the total amount of
StarLink corn in the food supply, while extremely low today, will
rapidly decline.
It is important to highlight the new information that has been provided
in the Supplemental Safety Assessment. In addition to new digestibility
results demonstrating that Cry9C is in fact digestible, the new
submission by Aventis contains two important new scientific studies.
The first is that Cry9C protein did not cause allergic reactions in
individuals who are allergic to other foods, such as soybeans, peanuts,
wheat, rice, milk, eggs and shrimp (which are clinically among the most
important food allergens present in the food supply). This study means
that Cry9C is not a cross-reactive allergen which could cause symptoms
in food allergy sufferers generally.
The second is a comprehensive new risk assessment that makes the
hypothetical assumption that Cry9C protein is, in fact, a potential
allergen. In this analysis, Aventis and Novigen Sciences show that even
if Cry9C is an allergen which is as potent as the most notorious food
allergen known, in peanuts, that insufficient Cry9C protein would be
present in foods to possibly elicit an allergic reaction. In other
words, if Cry9C is as potent as peanut allergens, there would still be a
significant safety factor due to the extremely low quantity of Cry9C
protein and the extremely low percentage of StarLink corn in the total
corn food supply. Peanut is used in the Aventis risk assessment as a
hypothetical and conservative basis for comparison. Remember, however,
that Cry9C is unlikely to be an allergen as potent as peanuts. Thus,
this worst-case scenario should be viewed as illustrative of the
reasonable certainty that StarLink corn presents no harm in the food
supply.
In addition BIO contracted with Medallion Laboratories to quantify the amount of Cry9C protein in finished products. The results of this study show that while the Starlink protein is detectable at the expected level in the grain, it is not detected easily in any of the other samples. During the masa process, discarded steep and wash water fractions were analysed and none of the target protein was detected. In the masa dough, a very (very) low level of the target protein was detected. This is at the limit of detection of the method, below the level that can be quantified reliably. At or below this extremely low level, accurate or reliable interpretation of the results is not possible. The limit of detection of the protein for the two western blot assays used is 50 picograms of protein in a single band. In the samples representing a dough process and a cereal process the level of the target protein was slightly higher, although still barely above the level of detection. Again this was not at a level that can be quantified without further extensive experimentation.
This study confirms the premise that the processing of corn containing the Starlink protein Cry9C, significantly reduces the level of detectable protein. This takes into account and allows for the challenges of extracting the protein from the complex matrix, which is produced in the samples during food processing.
The report from Medallion Laboratories is attached.
Given all this information, StarLink corn cannot be considered a potential allegen, especially under the circumstances of limited exposure. I urge you to grant a time limited exemption from the requirement of a tolerance for StarLink corn.
Executive Summary
The purpose of this study was to determine the effect of food processing on the Bacillus thuringiensis (Bt) produced protein Cry9C, which is the protein of interest in Starlink corn, to estimate the potential exposure of this protein in selected Starlink derived food products. Samples of Starlink corn and a conventional corn variety were treated identically through three simulated food-processing methods. All of these experiments were bench top experiments, which mimic and represent the processing steps used in various food manufacturing systems. The time allowed for this study did not allow method validation or rigorous reproduction of results, however the data has been duplicated by similar methods in two different laboratories and evaluated by a number of additional protein scientists. The food processing systems were "a bench top masa dough process", a rapid visco amylograph measurement representing a cereal process and an elevated temperature farinograph measurement representing a more extended cereal cooker/extruder process. Samples were taken at various points during these processes. Attention was focussed on the masa process, the ingredient used in taco shells and some corn chips.
Detection of protein was done using Western Blot analysis which allows for the most rigorous extraction and is a very sensitive protein-based method of detection. The more usual protein detection systems applied in food processing are not sensitive enough to detect the extremely low levels found with transgenic proteins. Nevertheless it was deemed appropriate to compare the Starlink variety with the conventional variety using capillary electrophoresis. As expected the Cry9C protein was not detected with this method in either the untreated seed or after boiling for 60 minutes in alkaline solution.
Further samples were analysed using Western Blot analyses. As there are a number of alternatives when using this technology, the two laboratories analysing the samples used two slightly different techniques.
The results of this study show that while the Starlink protein is detectable at the expected level in the grain, it is not detected easily in any of the other samples. During the masa process, discarded steep and wash water fractions were analysed and none of the target protein was detected. In the masa dough, a very (very) low level of the target protein was detected. This is at the limit of detection of the method, below the level that can be quantified reliably. At or below this extremely low level, accurate or reliable interpretation of the results is not possible. The limit of detection of the protein for the two western blot assays used is 50 picograms of protein in a single band . In the samples representing a dough process and a cereal process the level of the target protein was slightly higher, although still barely above the level of detection. Again this was not at a level that can be quantified without further extensive experimentation.
This study confirms the premise that the processing of corn containing the Starlink protein Cry9C, significantly reduces the level of detectable protein. This takes into account and allows for the challenges of extracting the protein from the complex matrix, which is produced in the samples during food processing.
OBJECTIVE
The questions to be answered in this study are: first, can the Starlink Cry9C protein be detected in the grain samples supplied; and second, when the grain is processed to simulate traditional food processes, can the protein be detected using the appropriate detection methods.
SAMPLES
Two samples of corn were obtained from Aventis. These samples were described as 100% Starlink and 100% Starlink free corn.
FOOD
PROCESSING METHODS
Three different food processes were simulated in this study.
Masa dough process - the dough was further processed to tortillas, and corn chips. See Appendix #1.
Cereal process - using the Rapid Visco Analyser (RVA) which is used in conventional food systems to predict or monitor the functionality of grain through a manufacturing process. It was felt that the closeness of this test to the temperature and mechanical forces that grain will undergo in manufacturing would represent most closely the changes in manufacturing. See Appendix #2.
Farinograph measurements are used commonly in understanding the performance of different grain cultivars in dough type systems. See Appendix #3.
MICROSCOPIC
ANALYSIS OF SAMPLES
Examination of samples in the masa process was done to ensure that the appropriate particle sizes and starch gelatinization stages were reached at the samples points.
In the treatment of grain in high alkaline solution and simmering for a minimum of one hour (nixtamalization process) the moisture is seen to thoroughly permeate the grain. The combination of moisture and heat treatment as indentified by the extent of starch gelatinization does not progress more than 7-10 cells below the pericarp level. See Figure #1. This is in agreement with that reported to us by a number of commercial operations.
The grain, after steeping overnight (minimum 12 hours), was ground in a food processor to a particle size that was easy to sheet for tortillas. The tortillas made from this dough had a similar texture to those purchased at the Mexican market. The wetness of these particles and clumping into dough made a direct measurement of size impossible. The starch gelatinization status in the masa dough did not change from that seen in the steeped grains.
PROTEIN
ANALYSIS
Capillary
Electrophoresis
To confirm the patterns and levels of traditional corn proteins in the samples capillary electrophoresis analysis was performed. See Appendix #4 and Figures 2 and 3.
The level of Bt Cry9C was below the level of detection of this method. Samples spiked with nanogram levels of purified Bt Cry9C protein provided by Aventis were also not detected.
Western
Blots
The Western Blot analysis can be done with a number of alternative procedures. In this study the samples were analysed using two slightly different methods, including different extraction buffers (one representing strong extraction, the other a milder method).
In the milder extraction method all samples were
extracted with deionized water by vortexing 1 g of sample with 4 mL of
water for one minute. In cases where sample size was other than 1 g the
same ratio of sample to water was kept. After vortexing, a one-fourth
volume of 1M Tris/100 mM EDTA, pH 7.6 was added. Samples were then centrifuged at 10,000 rpm (Eppendorf Model 5804R centrifuge) for 10 min at 10 C. 75 µl of supernatant liquid from each sample were mixed with 25 µl of 4X NuPAGE (TM) sample buffer and 10 µl of 100 mM DTT and then heated to 85 C for 5 min. See Appendices #5 & 6.
When loading the gels, every attempt was made to put equivalent amounts of extracted protein onto the gels. This allowed a comparative qualitative analysis of the gels to be made but without further testing, a quantitative result cannot be obtained.
These analyses were completed with appropriate controls to show the size of the proteins to be detected, the ability of the method to extract the protein from the matrix and to ensure no cross reactivity with other corn proteins.
The antibody used for this analysis was provided by Strategic Diagnostics Inc.
This polyclonal antibody is different than the monoclonal antibody used in the ELISA test in that it will react with denatured as well as native proteins. Preliminary blots showed strong bands in non-Starlink samples. This cross reactivity with non target proteins was eliminated by reacting the antibody with non-Starlink corn prior to using it in the analysis. The Western Blots that have been blocked are indicated on the Figures.
ELISA
Analyses
The ELISA analysis uses specific antibodies to locate the target protein. The Bt Cry9C ELISA kit used in this analysis was provided by Strategic Diagnostics Inc. This test uses both monoclonal antibody and polyclonal antibodies. It does not react positively with a Western Blot and is therefore unable to detect the target protein once it has been denatured. Kit instructions were followed.
Lateral Flow
Strip Analysis
The strip test is a rapid method for detection of the Cry9C protein. This test also provided by Strategic Diagnostics Inc, uses the same monoclonal antibody from the ELISA test. See Appendix # 7.
RESULTS AND
DISCUSSION
The varieties of corn representing Starlink and non-Starlink were confirmed using both Lateral Flow strips and ELISA analysis.
The simulated food processes used in this study are well understood in food research laboratories. It is not the purpose of this report to dwell on this except to say that many commercial manufacturing processors use these methods to predict the functional properties of grain. They can be used to maintain processing conditions for optimum cook systems or to troubleshoot problems encountered in manufacturing systems with changes in incoming grain supplies. We believe that they provide an appropriate and reliable basis to simulate the effects of processing on the Cry9C protein in grain.
The processing of food with heat might be expected to denature the protein and this was observed when the protein could not be detected in the cooked grain using the SDI lateral flow strip and the ELISA test. It was noted that the number of high molecular weight proteins (observed in silver stained gels, Figure # 4) decreased significantly with the different cooking processes. It was not possible to identify the breakdown products of these proteins.
Masa
Process
The masa process produced a soft grain that separated from the pericarp fraction easily. The dough process created a soft, damp, cohesive mass that was broken into smaller pellets when sampled. Several attempts to simulate the masa flour were made, but replicating the structural effects of the very high heat process was difficult. It was expected that the "flow of the starch in the popping process" would further perturb the corn proteins, but with the limited time available for this study it was decided not to pursue this challenge at this time.
Samples of the cooking waters, rinse waters and steep waters were retained for testing. There was no detectable Cry9C in these fractions using the ELISA or strip tests. Western Blot analysis was also negative. See Figure # 5. Key to Figure #5 is Table #1.
Samples of the masa dough were extracted and the resulting protein fraction was tested for the presence of Cry9C protein. This analysis was repeated in both laboratories and similar results were obtained. See Figures # 6a & 7 and Table #2.
Similar samples of the RVA dough and the Farinograph dough were also extracted. The additional processing and smaller particle size (and released starch granules) in the Farinograph samples caused significant protein extraction problems. In fact, sufficient protein for analysis was only obtained after digestion of the sample with amylase. The heat and mechanical action of this test have denatured many of the proteins of the grain. It is expected that the denatured proteins will interact with the starch molecules that are released by gelatinization of the starch and recombination of both proteins and starch will occur as the sample is cooled. The samples showed levels of Cry9C protein at the level of detection for this method. This detection level is calculated to be in the order of 50 picograms per single band. The low level of protein in these samples does not allow for quantification by this method. See Figures # 6b & 7 and Table #2.
CONCLUSION
The results of this study reconfirms that the level of the transgenic Bt protein, Cry9C in Starlink corn is many orders of magnitude lower than the other proteins found in corn. It further shows that any type of heat processing denatures the protein such that it is not identified by the ELISA method used in this study. Further processing significantly reduced the amount of protein that could be detected using the Western Blot analysis. In fact it is barely detectable by this method.
ACKNOWLEDGEMENTS
The following people contributed to this study in many and various ways. Their assistance is appreciated.
Ray Shillito, Aventis
Dale Onisk, Strategic Diagnostics Inc
Jim Stave, Strategic Diagnostics Inc
Kim Magin, Monsanto Co
Dave Grothaus, Pioneer Hi-Bred Intl
Charles Shelburne, 3M
Figures nos. 5, 6a & 6b were provided by Stategic Diagnostics Inc.
APPENDIX 1
STARLINK CORN
COOKING PROCEDURES
NIXTAMALIZATION
Weigh 1000 g of raw dried dent corn into a 4L stockpot. Add 1000 mL of deionized water. Stir for 2 minutes.
Take 50 mL of supernatant fluid. SAMPLE A. Add 12.5 mL Tris/EDTA buffer to this sample to stabilize. Refrigerate for further analysis.
Add 10 g Ca(OH)3 to the corn pot Stir for 2 minutes.
Take 50 mL of supernatant liquid. SAMPLE B. Add 12.5 mL Tris/EDTA buffer to stabilize. Refrigerate.
Heat corn to boiling uncovered.
When boil first occurs, take 50 mL SAMPLE C. Add 12.5 mL Tris/EDTA buffer to stabilize. Refrigerate.
Continue to boil corn for 1 hour, replenishing water continuously to keep kernels covered.
After 1 hour boil, take 50 mL supernatant as SAMPLE D. Add 12.5 mL Tris/EDTA buffer to stabilize. Refrigerate.
Remove corn from heat and allow to steep 12 hours. At end of steep, take 50 mL supernatant as SAMPLE E. Add 12.5 mL Tris/EDTA buffer to stabilize. Refrigerate.
RINSING
Rinse the corn with cold tap water in a colander three times. Rub the kernels between hands to remove the pericarp. Put the corn back in the pot and allow the tap to run overflowing into the sink until the water is clear and free of floating pericarp. Drain in the colander.
DOUGH
Put the corn in a large food processor and grind until a flour consistency. Turn off the processor and scrape sides frequently. For 1 Kg of corn, use approx. 200 mL of additional water to make the dough. Continue processing for approx. 10 min to get a dough that is consistent and not lumpy. Close inspection of the dough will show very fine granulation, with very few large pieces of horny endosperm remaining. Be careful when adding water: if the dough gets too sticky, it will not process. Add just enough water so that a large doughball forms and moves around the bowl with the knife slicing through it. If the dough gets too wet, remove it from the bowl and spread it out on the counter to dry.
When finished, dough will be easy to work, and not sticky. Place in a plastic bag and refrigerate. LABEL AS SAMPLE F.
TORTILLAS
If dough has been refrigerated for a long time, it will be drier than optimal. Too dry, the dough will crumble, too wet and it will be sticky. Add water to proper consistency.
Roll a small bit of dough between hands to form a perfect sphere. Put a square of waxed paper on the bottom plate of the tortilla press. Place the ball of dough on the press. Cover with another square of waxed paper. Press the dough in the press to a uniform thickness, approx. 2 mm thick. Remove from the press and waxed paper. Judge moisture content of dough - too wet will be impossible to remove from waxed paper, too dry will not form perfect circle, but will serrate at edges.
Place the formed tortilla on a hot 400 F griddle. Allow 1-2 min to cook per side. When done, the tortilla will be slightly browned on the outside, and the inside will puff up, separating the 2 sides and steaming the inner dough.
Remove from the griddle and label as SAMPLE G.
BAKED CORN
CHIPS
Cut the tortillas into wedges. Place on a towel and let dry for 30 minutes in RT air. Place on a cookie sheet and bake in a 400 F oven for 10 minutes. Cool. LABEL AS SAMPLE H.
FRIED CORN
CHIPS
Cut the tortillas into wedges. Place on a towel and let dry for 30 minutes in RT air.
Fry in vegetable oil for 1-2 min at 400 F until they just start to turn brown. Remove from oil and place on a paper towel to drain.
LABEL AS SAMPLE J
APPENDIX 2
Rapid Visco Analyser
(RVA) Procedure:
Use a Computrac calibrated to the sample matrix to obtain a sample moisture.
Place a plastic weigh boat on the balance, and press the tare bar. Find the proper sample weight adjusted for moisture content from the appropriate sample weight table.
Weigh the proper amount of sample into the weigh boat.
Place an aluminum sample canister on the balance and press the tare bar.
Weigh the correct amount of distilled water to the canister as found on sample weight table.
Adjust the distilled water weight to within .02 of the correct value with a disposable dropper.
Transfer the ground sample to the canister containing the distilled water. Use a lab spatula to aid the procedure. Check the total weight to make sure it matches the total weight as shown on the sample weight table.
Place the impeller into the sample canister. Twist the impeller to the right one full turn, and then back to the left one full turn.
Scrape the side of the canister with the blade of the impeller to be sure that all of the sample is submerged.
Place the top of the impeller into the sample coupling on the RVA. The blue cut-out notch should be positioned towards you. Slide the impeller into the coupling.
Spin the coupling to ensure the sample canister is positioned correctly. There should be no visible or audible signs of friction.
Place both hands on top of the blue tower. Push straight down with a slow, steady motion. The tower will lock and begin to spin. The RVA will now run to the end of the test.
|
Elapsed Time |
Temp |
|
Idle Temp |
25.0 C |
|
0-2 min |
25.0 |
|
2-6 min |
Ramp to 97.0 |
|
6-10 |
Hold at 97.0 |
|
10-11 |
Cool to 25.0 |
|
11-20 |
Hold at 25.0 |
APPENDIX 3
FARINOGRAPH
METHOD
Samples of StarLink and Non-StarLink corn were ground and cooked in a Farinograph (Brabender Instruments, Inc) for 15 minutes at 95 C. Samples were weighed to constant moisture method of 60g sample plus 25 mL water assuming corn at 12% moisture. Actual sample weights were changed based on a Computrac moisture measurement.
Apparatus:
Brabender Do-Corder E330 C. W. Brabender Instruments Inc.
50 East Wesley Street
equipped with a 2-16-000 PO Box 2127
mixer/measuring head (blade South Hackensack, NJ 07606
speed ratio, drive to (201)343-8425
driven: 3:2) with Sigma-type
blades, Type SB
Instrument
Settings:
1. The Test Speed is 100 rpm.
2. Sensitivity Selection is 1:5.
3. Zero Suppression is set to 0.
4. The oil bath controlling the measuring head mixing bowl temperature is set to 95 C. The temperature of the bowl as measured by a thermometer in the thermometer port of the bowl will be 94.5 to 95 C.
5. The sample size is 60 grams of corn flour and 25 mLs of distilled water assuming an corn flour moisture of 12% WB. Obtain a least 130 grams of sample and determine the sample moisture with a primary or certified secondary method.
APPENDIX 4
Capillary Electrophoresis of Corn Proteins
Background
Capillary electrophoretic methods are a family of separation techniques in which separations are achieved by the differences in migration rates for sample components in an electric field applied to a capillary tube filled with an electrolyte. The presence of an electric field causes the movement of sample ions by electrophoretic migration and bulk flow of the electrolyte solution by electroosmosis. In capillary gel electrophoresis ions are separated by their electrophoretic mobility through a porous gel network.
As with conventional SDS-gel electrophoresis, capillary SDS gel electrophoresis is utilized for size separation of proteins and the technique is based on the phenomenon that SDS binds to proteins in a constant ratio. The constant mass-to-charge property of the SDS-bound proteins allows separation according to differences in protein molecular weights. The staining of conventional slab SDS- gels, which allows the eye to serve as a detector, is replaced in Capillary Electrophoresis by an on-line detector (in this case UV). Limit of detection (LOD)for CE-SDS-Gel Electrophoresis is 1 microgram per microliter however, minimum volume of solution for CE injection is 500 microliters
CE
Analysis
Capillary: 47cm x 100 micrometers i.d. (SDS gel-filled)
Voltage: 14.1 KV
Buffer: 0.12M Tris/HCl/1%SDS, pH 6.6
Temperature: 25 C
Injection: 30 seconds
Detection: UV 214 nm
LOD: 1mcg/microliter
CE CodeSample
Description
Crybln20a Blank (contains positive marker protein)
Crypn20b Cry9C standard (contains positive marker protein) - protein not detected
Cry1dn20a Non-Starlink Corn after boiling for 60 minutes
Cry2dn20a Starlink Corn after boiling for 60 minutes
Results
The electrophoreograms indicate the presence of traditional corn proteins in both Starlink and Non-Starlink corn samples. See Figures 2 & 3. At the CE limit of quantitation/limit of detection, Bt Cry 9C protein was not detected in the standard or in the Starlink corn.
APPENDIX 5
SDS-PAGE and Western Blot Analysis of Cry9c
Samples
21-Nov-00
1. Samples are prepared in 4X NuPAGE sample buffer with DTT (Invitrogen).
2. The samples are heated (75C) for five minutes and cooled to room temperature.
3. 4-12% Bis-Tris- gel(s) (10 wells, 1.0mm thick, set up in Novex SureLock Mini Gel system (Invitrogen).
4. To each well 15-20 µL of sample was added
5. To two wells 5 µL of Molecular Weight standard was added (Novex MultiMark pre-stained standards).
6. Gels run at 200V constant for about 30 minutes or until dye front reaches end of the gel.
7. After the run the Gel was immediately transferred to a Novex Xcell Western blot module, as appropriate.
8. The Novex western blot system was used according to the manufacturer’s instructions.
9. Transfer to 0.45 µm nitrocellulose (Bio-Rad Cat. # 162-0145) was at 30V constant for one hour.
10. Membrane was blocked with 5% Non Fat Dried Milk (NFDM) in Tris Buffered Saline (TBS), pH 8.0 overnight at 4C.
11. Membrane was immuno-blotted with Rabbit Anti-Cry9c purified antibody.
12. The Rabbit antibody was added at 10µg/mL in 1% NFDM in PBS, pH 8.0 (10-15 mL).
13. Membrane was incubated for 1 hour at Room Temperature with shaking on an orbital shaker.
14. Membrane was washed for 30 minutes with TBS, 0.05% Tween 20.
15. To blot add 15 mL AP-Goat anti-rabbit IgG (H+L) (Zymed, Cat.# 62-6122) at 1:1000 in 1% NFDM in PBS, pH 8.0.
16. Incubate for 1 hour at RT with shaking.
17. Wash for 30 minutes with TBS, 0.05% Tween 20.
18. Add 10 mL BCIP/NBT substrate (Sigma, Cat. # B-5655) to blot until bands develop.
19. Stop reaction by rinsing membranes with distilled water.
APPENDIX 6
SDS-PAGE and Western Blot Analysis of Cry9c
Samples
13-Nov-00
1. Samples are prepared in Laemmli sample buffer with 2-ME (Bio-Rad Cat. # 161-0737) :
2. The samples are boiled for five minutes and cooled to room temperature.
3. 4-15% Tris-HCl gel(s) (12 wells, 20 µL capacity, Bio-Rad, Cat. # 161-1176, Exp. 02-Feb-01) was setup in Bio-Rad’s Mini Gel system.
4. To each well 15-20 µL of sample was added
5. To two wells 8 µL of Molecular Weight standard was added (we use Novex SeeBlue and MultiMark pre-stained standards).
6. Gel are run at 125V constant for about 1 hour or until dye front reaches end of the gel.
7. After the run the Gel was rinsed with Tris-Glycine buffer (Transfer buffer (Bio-Rad Cat.# 161-0734)) for about 60 minutes with one change of buffer.
8. distilled water for about 30 minutes.
9. The Bio-Rad’s mini Transblot system was used (we follow the manufactures instruction).
10. Transfer to 0.45 µm nitrocellulose (Bio-Rad Cat. # 162-0145) was at 100V constant for one hour.
11. Ponceau S solution (Sigma) (5ml) was added to the blot, and prominent bands were marked for reference.
12. Membrane was blocked with 5% Non-Fat Dried Milk (NFDM) in Tris Buffered Saline (TBS), pH 8.0 overnight at 4C.
13. Membrane was immuno-blotted with Rabbit Anti-Cry9c purified antibody.
14. The Rabbit antibody was added at 10µg/mL in 1% NFDM in TBS, pH 8.0 (10-15 mL).
15. Membrane was Incubate for 1 hour at Room Temperature with shaking on an orbital shaker.
16. Membrane was wash for 30 minutes with TBS, 0.05% Tween 20.
17. To blot add 15 mL AP-Goat anti-rabbit IgG (H+L) (Zymed, Cat.# 62-6122) at 1:1000 in 1% NFDM in TBS, pH 8.0.
18. Incubate for 1 hour at RT with shaking.
19. Wash for 30 minutes with TBS, 0.05% Tween 20.
20. Add 10 mL BCIP/NBT substrate (Sigma, Cat. # B-5655) to blot until bands develop.
21. Stop reaction by rinsing membranes with distilled water.
TABLE 1
SAMPLE KEY TO FIGURE 5
|
Sample Identifier |
Corn Source |
Process Step |
|
1A |
StarLink Free |
Sample of water after adding to corn
stirring 2 min |
|
2A |
StarLink |
Sample of water after adding to corn
stirring 2 min |
|
1B |
StarLink Free |
Sample of water after addition of Ca(OH)2 |
|
2B |
StarLink |
Sample of water after addition of Ca(OH)2 |
|
1C |
StarLink Free |
Sample of water at first boil |
|
2C |
StarLink |
Sample of water at first boil |
|
1D |
StarLink Free |
Sample of water after boiling 1
hour |
|
2D |
StarLink |
Sample of water after boiling 1
hour |
|
1E |
StarLink Free |
Sample of water after 12 hour
steep |
|
2E |
StarLink |
Sample of water after 12 hour
steep |
TABLE 2
StarLink Cry9C
Protein Analysis Summary
|
Sample Identifier |
Description |
Flow Strip |
ELISA |
Western Blot Lab1 |
Western Blot Lab2 |
|
1 |
StarLink-Free Raw Corn |
NO |
NO |
- |
NO |
|
2 (or C+) |
StarLink Raw Corn |
YES |
YES |
YES |
YES |
|
1F |
Masa Dough
StarLink-Free |
NO |
NO |
NO |
NO |
|
2F |
Masa Dough
StarLink |
NO |
NO |
YES |
YES |
|
1G |
Tortilla
Starlink-Free |
- |
- |
NO |
- |
|
2G |
Tortilla
Starlink |
- |
- |
YES |
- |
|
1H |
Baked Chips
StarLink-Free |
- |
- |
NO |
- |
|
2H |
Baked Chips
StarLink |
- |
- |
YES |
- |
|
1J |
Fried Chips
StarLink-Free |
- |
- |
NO |
- |
|
2J |
Fried Chips
StarLink |
- |
- |
YES |
- |
|
1K |
RVA Cook
StarLink-Free |
- |
- |
NO |
NO |
|
2K |
RVA Cook
StarLink |
- |
- |
YES |
YES |
|
1L |
Farinograph
StarLink-Free |
- |
- |
NO |
NO |
|
2L |
Farinograph
StarLink |
- |
- |
YES |
NO |
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