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KENDRICK LABS CATALOGPrint CatalogCall 800-462-3417 or email 2d@kendricklabs.com for a price quote or to receive printed information by mail.2D ELECTROPHORESISMK-1 Mailing Kit. This kit contains ten 1 ml tubes of Urea Sample Buffer, ten 1 ml tubes of SDS Boiling Buffer; ten 1 ml tubes of SDS Boiling Buffer minus BME; ten 1 ml tubes of Osmotic Lysis Buffer; ten 100 ul tubes of 10X Nuclease Stock; and two 100 ul tubes of Protease Inhibitor Stock Solutions. The compositions of these solutions is given in the Kendrick Labs booklet "Suggestions for Sample Preparation". In addition a Styrofoam mailer, inner carton for radioactive samples and mailing instructions are included. Price: $195 includes shipping on dry ice. 2D-ES-1: 2-D ELECTROPHORESIS BASIC SERVICE.
Our standard gel
size is perfect
for immunoprecipitations, subcellular fractionations, most bacterial
preparations and many proteomics
applications. One Coomassie blue stained, dried 2-D gel of dried
dimensions 16 cm wide x 13 cm long, is returned for each sample along with a pH
gradient plot and a method description sheet suitable for reports or
publications. The example below is from an Escherichia
coli sample prepared in our Urea sample buffer (left) and SDS Boiling
Buffer (right) run identically on a pH 4-8 IEF gradient and 10% acrylamide slab
gel. The great advantage of SDS buffer is ease of sample preparation. Molecular weight standards (220,000, 94,000, 60,000, 43,000,
29,000, and 14,000) appear as bands on the right side of the gel labeled with
press-on numbers. One isoelectric point marker, added to each sample as an
internal standard, is marked with an arrow. Our standard IEF tube gel contains
2% pH 4-8 BDH ampholines; transparency drying (STP-3) is standard. The duplicate
gel rate is used for duplicate gels with different stains and also for blots,
but not for different gel conditions. More photos of example gels.
See 2D-ES-5 for our large format option.
CUSTOM PH GRADIENTS are available using ampholines of pH 3.5-10, 2.5-5, 4-6 and 7-9. For basic proteins with pI's greater than 9, non-equilibrium pH gradient electrophoresis is used (NEPHGE, O'Farrell, P. et. al. Cell 12: 1133, 1977). Buffers and electrodes are reversed and the pH gradient is created using pH 8-10.5 ampholines. Indicate which custom pH gradient you need on the back of the sample ID form. (No extra charge) 2D-ES-2: 2D ELECTROPHORESIS WITH PEPTIDE SLAB
GELS. The 16.5% peptide slab gels of Schagger and von Jagow (Anal. Biochem.
166: 368, 1987) are useful to resolve low MW proteins (3,000 - 14,000). In
general it is better to load low MW proteins heavily (10 ug/spot). Note
that ampholines may interfere in the MW range 2,000-6,000. 2D-ES-3: NATIVE 2D ELECTROPHORESIS For
native 2D the urea and NP-40 are omitted from the IEF tube gel; SDS and BME
are omitted from the 2nd dimension slab gel. No denaturing agents are used
in the sample buffer. Many proteins are insoluble under these conditions and
don’t resolve. However, occasionally it’s useful for soluble proteins in
serum and CSF. 2D-ES-4: 2-D ELECTROPHORESIS WITHOUT
SULFHYDRAL REAGENT Same as 2D-ES-1 except
2-mercaptoethanol and dithiothreitol are omitted from sample buffer,
internal standards, and all 2-D solutions. 2D-ES-5 LARGE FORMAT 2D
ELECTROPHORESIS The larger size gives better resolution for
proteomics applications; heavier loads may be applied to obtain more
material for mass spectrometry. One Coomassie blue-stained, dried 2D gel
of dimensions 20 cm wide by 22 cm long is returned for each sample along
with a pH gradient plot and a method description sheet. Our
standard IEF tube gel contains 2% pH 4-8 BDH ampholines. Molecular
weight standards (220, 94, 60, 43, 29, and 14 kDa) appear as bands on
the right side of the gel labeled with press-on numbers. One IEF marker,
added to each sample as an internal standard, is marked with an arrow.
Transparency drying (STP-5, page 13) is standard. See above for Custom
pH gradients. Typical protein loads are 100 (silver staining) or 600 ug
(Coomassie blue) in 25-150 ul. The example shown below is a silver stained LF gel, see STP-2LF for silver staining.
2D-ES-6: Blue Native
Polyacrylamide Gel Electrophoresis (BN-PAGE)
is a charge shift method developed by Schagger and von
Jagow (Anal. Biochem. 199: 223-231, 1991) for isolation and
characterization of large multi-protein complexes (MPCs) in their native
state. Binding of Coomassie blue G-250 in the absence of denaturing
detergents allows for first dimension separation according to the size
and shape of the complex on 4-13% gradient gels. Coupling first
dimension BN-PAGE with a second dimension of SDS-PAGE allows for the
dissociation of individual subunits based on molecular weight. Further
analysis by Western blotting or mass spectrometry can be performed to
identify specific subunits within MPCs. We currently offer both BN-PAGE
as the first dimension only, or BN-PAGE followed by SDS-PAGE as a second
dimension. BN/SDS PAGE has proven a valuable tool for studying the
functional proteomics of mitochondrial protein complexes (Methods 26:
327-334, 2002), membrane protein complexes (Anal. Biochem: 217: 220-230,
1994), and whole cell lysates (Molecular and Cellular Proteomics: 3.2:
176-182, 2004). Please call to discuss before sending samples for BN-PAGE.
2D-ES-7: pI MARKERS - CARBAMYLATED CARBONIC ANHYDRASE (MW 29,000) and/or CARBAMYLATED CREATINE PHOSPHOKINASE (MW 40,000).
These proteins, purchased from Pharmacia and calibrated by Kendrick Labs, are added as internal pI standards for
2D gels. A pH gradient standard curve may be determined from the train of charge isomers
(shown in the figure below) for calculation of pI's for unknown proteins. Note that these pIs are not absolute values but are for conditions of 9 M urea and
22oC. Price: Add $16 per gel.
2D-ES-8: PARTIAL SAMPLES. Corresponding tube gel sections from different tubes are run on a single
2D slab gel as shown in the figure below. This is especially useful for obtaining more protein for sequencing since fewer
gels are required.
2DE/Top Mass Spectrometry Staining Blotting Radioactivity Sample Preparation Misc 2D-ES-9: MANUAL COMPARISONS of 2D PATTERNS FOR
DIFFERENCES. We find that HBC (Human Brain Comparison)
is a reliable method for finding changes in 2D patterns. In this method two
experienced analysts independently compare stained gel pairs on a light box for
differences. Results are presented as color coded outlines of spots on
transparent overlay covering the gels. Free duplicate gels are run to
confirm differences for samples scheduled for manual comparisons. 2D-ES-10: COMPUTERIZED COMPARISONS
OF 2D GEL PATTERNS FOR DIFFERENCES. This analysis includes laser
scanning and computerized analysis with SameSpots software from Non-Linear
Dynamics. Requires prior 2D electrophoresis but free duplicate gels are run
and analyzed to confirm differences for samples scheduled for computerized
comparisons. Generally 400-800 spots are quantified per Standard Format gel;
800-1200 spots are quantified per Large Format gel. Spot density values are
expressed as spot percentages (individual spot density as a percentage of
total density in all spots analyzed) to normalize for differences in sample
loading or staining. Results are presented in a complete report including a
summary table showing spot number, pI, MW, ratios (fold difference) and p
values as a second measure of difference. The final report also includes
figures of images showing numbering, montage images for every differing
protein spot, methods and pH gradient plot. A CD containing data and image
files is included in the package. Note that our price includes licensing
fees paid to Nonlinear of $50/gel image. 2D-ES-11 DATABASE
MANAGEMENT. This archiving service allows pattern comparisons
across sets of experiments by matching protein spots in new
experiments to corresponding spots on a master gel used in a
previous experiments. Thus corresponding spots have the same numbers
across experiments. This is important in many situations, for example cell
lines that are differentially manipulated as your research progresses, but
less important in others, for example when a few key proteins are changing
and may be matched by eye across experiments. Samples must have a high
degree of similarity for database management which is often much more
difficult than expected. 2D-ES-12 QUANTIFICATION OF PROTEINS RESOLVED BY 2-D ELECTROPHORESIS. For clients knowing which proteins need to be quantified. Scanning (2D-ES-13) is included. Data is reported in integrated spot density in density units,
or as a percentage of total image density. 2D-ES-13: LASER DENSITOMETRY. Stained gels and
x-ray films are digitized with our laser densitometer from Molecular
Dynamics. The densitometer is linear over the range 0-3.0 optical density
units as verified by calibrated filters. Narrow O.D. ranges can
be expanded to full scale (8 bit, 0-255) if required.. Scanning resolution
is 100 microns/pixel; the final black & white tif images are 3.3 Mbytes each
for standard format and 7 Mbytes for large format. 2D-ES-14: ELECTRONIC PHOTOS AND FILES. The 10 mB
black & white files obtained from laser densitometry are unsuitable for
documents and the web. Our Afga Arcus II and HP 1200 scanners gives
excellent color images in tif format that are useful for publication and
emailing (jpg format). The price includes tif and jpg image files on a CD
mailed with the dried gels, and jpg images emailed to provide previews of 2D
patter ns. 2DE/Top Comparisons Staining Western blotting Radioactivity Sample Preparation Misc Protein Identification by Mass Spectrometry We have considerable experience in sending 2D gel proteins to university and commercial core facilities for identification by mass spectrometry fingerprinting (also called peptide mass matching) and for amino acid sequencing. Turnaround time for mass spectrometry is usually about two weeks. We highly recommend Columbia University Protein Core Facility http//cpmcnet.columbia.edu/dept/protein Contact Dr. Mary Ann Gawinowicz, tel: 212-305-3631, email: mag4@columbia.edu 2D-ES-19 Spot cutout for mass spectrometry or sequencing We do this manually and are obsessively careful. The cutout spots are placed in Eppendorf tubes for express mail to the core facility of your choice; our recommended facility is Columbia University. The cost includes a letter to the core listing the spots, plus a copy of the letter for you along with an image, photocopy or original gel showing spot locations. We make sure that each spot has a unique identifier. Price: $16 per spot. 2D-ES-15 MASS SPECTROMETRY FINGERPRINTING to identify proteins from Coomassie blue or special silver stained gels. The method is at least 10 times more sensitive than sequencing by Edman degradation and less expensive, but the protein must be in the databases. Proteins are digested with an enzyme of known specificity, usually trypsin, and the resulting peptide mixture, without separation, is analyzed by MALDI (matrix-assisted laser desorption ionization) MS. The peptide masses obtained by MS are entered into a search program which scans the database, NCBI or Genpept, to find a match. This method will not produce sequence data from the protein, only a unique set of peptide masses enabling identification. The mass spectrum will usually give 25-60% sequence coverage (number of amino acids in identified peptides/number of amino acids in whole protein). Search programs used at the Columbia Protein Core Facility are ProFound at http://prowl.rockefeller.edu/profound_bin/WebProFound.exe and MS-Fit at http://prospector.ucsf.edu. References for
peptide mass mapping 2. Mann, M., Hojrup, P., and Roepstorff, P., Biol. Mass Spectrom. 22:338-345 (1993). 3. Cottrell, J.S., Pept. Res. 7:115-123 (1994). 4. Shevchenko, A., Jensen, O.N., Podtelejnikov, A.V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., and Mann, M., Proc. Natl. Acad. Sci. U.S.A. 93: 14440-14445 (1996) Price for 2D-ES-15: $290 per polypeptide spot including database searching by hand, and invoicing through Kendrick Labs. 2D-ES-18 MASS SPECTROMETRY SEQUENCING
(LC/MS/MS) to identify proteins or to determine the unknown amino
acid sequence of peptide fragments of the protein of interest cut from
Coomassie blue or special silver stained gels. This method is
recommended for spots or bands which might contain mixtures, for spots
which could not be identified by MALDI, and for samples from species
with incomplete genomes. It is not affected by N-terminal blockage.
Protein spots are cut from Coomassie blue or special silver stained gels
and digested with trypsin. The digestion mixture is separated on an LC
Packings nano-lc on which the detector outlet is connected directly to
the nanospray source of a Micromass Q-Tof mass spectrometer. Peptides
are eluted at a flow rate of 200 nl/min and are scanned as they enter
the source. When a peptide is detected, the Q-Tof is programmed to
switch to MS/MS mode, which means that the eluting peptide is fragmented
by means of an applied collision energy and the resulting ions scanned
for several seconds. When the programmed time for MS/MS is done, the Q-Tof
switches back to MS mode and resumes scanning the eluting peptides.
Because peptides fragment at the peptide bond in a predictable way, the
fragmentation pattern can be used to deduce sequence information. The
ions resulting from fragmentation along with the mass of the intact
peptide can be used to search a database and identify the protein.
Proteins which show a good MALDI pattern but cannot be identified by
Mass Spectrometry Fingerprinting (2D-ES-15) are candidates for
identification by LC/MS/MS. Generally, LC/MS/MS is not more sensitive
than MALDI, but the data is acquired differently and can be more
informative.
2D-ES-20 PHOSPHORYLATION SITE IDENTIFICATION BY MASS SPECTROMETRY.
We
outsource this state-of-the-art service to the experts at the Laboratory
for Proteomic Mass Spectrometry at the UMass Med School. See:
www.umassmed.edu/proteomic/Leszyk/index.aspx for more information.
Contact Dr. John Leszyk, Tel: 508-856-7533 Email:
John.Leszyk@umassmed.edu
Note that
the success of phosphoprotein analysis depends on the type of protein
and the extent of phosphorylation. In some cases additional work may be
required.
2D-ES-16
INTERNAL AMINO ACID SEQUENCING BY EDMAN DEGRADATION.
An
enzymatic digestion is done on protein spots cut from Coomassie blue
stained gels followed by HPLC and sequencing of fragments. At least
10 pmoles is required. Usually the protein must be a reasonable dark
spot on a Coomassie blue stained gel; multiple spots are usually necessary
for proteins with MW greater than 60,000. Mass spectrometry
fingerprinting on 5-10% of the digestion sometimes allows identification
of the protein. In this case sequencing is not required but the cost
is $390 per spot because of the additional HPLC separation of
peptides. Sequencing by Edman degradation usually yields 12-20 amino
acids, enough for an oligonucleotide probe.
2D-ES-17
N-TERMINAL AMINO ACID SEQUENCING by Edman degradation is typically done after 2D
electrophoresis and PVDF transblotting to determine if a protein is
clipped. The protein must be loaded
heavily enough to see with Coomassie blue on the blot; the N-terminus
cannot be blocked. Note that bacterial proteins are seldom blocked
at the N-terminal while mammalian proteins are often blocked, up to 60-70%
of the time. 2D Gels/Top Comparisons Mass Spectrometry Blotting Radioactivity Sample Preparation Misc STAINING/DRYING PROCEDURESSTP-1:
COOMASSIE BLUE STAINING. We use Coomassie brilliant blue
R250 as our standard staining technique (O'Farrell, P. J. Biol. Chem. 250:
4007, 1975). This method involves an alcohol/acetic acid fix, an acetic acid
rehydration and an acetic acid destain. Generally, 1 µg of purified protein
gives a nice spot on a 2D gel (see our internal standard) and 0.1 ug is visible. Although not as
sensitive as silver staining, this method gives quantifiable results. (Burgess-Cassler
et. al. Clin. Chem. 35: 2297, 1989.) The linear range for plots of stain
density versus ng protein is broad and the method is reproducible. This stain
is compatible with mass spectroscopy.
STP-2: SILVER STAINING. Our silver stain is based on the ammoniacal silver/formaldehyde
method of Oakley et. al. (Anal. Biochem.105: 361, 1980.) This method
involves preliminary glutaraldehyde treatment of the slab gel to fix proteins by
cross linking. The pre-treatment also adds glutaraldehyde side chains to the
proteins, increasing sensitivity since these groups are sites for silver
deposition (Dion A, and Pomenti A, Anal. Biochem. 129: 390, 1983). We
find this method to be about 10 times more sensitive than Coomassie blue
staining, depending on the protein, even though less protein is loaded.
Generally, 50 ng of purified protein gives a highly visible spot on a 2D gel
(see our internal standard). However, some proteins that are detectable with
Coomassie don't stain at all with silver. Although very sensitive, this method
gives qualitative rather than quantitative results. The linear range for plots
of stain density versus ng protein varies widely from protein to protein and
also because day to day variability in stain and background intensity is
observed. Silver staining is not compatible with mass spectroscopy
fingerprinting. STP-3: SPECIAL SILVER STAINING FOR MASS
SPECTROSCOPY ANALYSIS. This special
silver stain (O’Connell and Stults, Electrophoresis 18: 349-359, 1997) omits the
glutaraldehyde step and is compatible with mass spectroscopy fingerprinting. It often negatively stains
proteins and so is not appropriate for computerized analysis. However, the pattern can be matched to our regular stain without
difficulty. Note however, that silver deposition interferes with the enzymatic
digestion required for mass spectrometry for most mammalian proteins. If possible use the less sensitive but
more reliable Coomassie stain. STP-4: SYPRO RUBY FLUORESCENT STAIN.
This Molecular Probes stain fluoresces linearly with protein over a range of
100-1000 ng and is compatible with mass spectrometry. See 2D-ES-13A
Fluorescent densitometry for imaging. STP-5: PRO-Q DIAMOND PHOSPHOPROTEIN FLUORESCENT STAIN. This Molecular Probes stain is used for detection of phosphoproteins and also is compatible with mass spectrometry. See 2D-ES-13A Fluorescent densitometry (page 9) for imaging. Price: STP-5 Standard Format Add $105 per gel; STP-5LF Large Format gels add $155 each. STP-6: EMERALD GLYCOPROTEIN FLUORESCENT STAIN
This Molecular Probes stain is specific for glycoproteins and also is
compatible with mass spectrometry. See 2D-ES-13A Fluorescent densitometry
(page 9) for imaging. STP-7: INVITROGEN PHOSPHOPROTEIN STAIN This new
stain is under development, We expect to offer it soon. See 2D-ES-13A
Fluorescent densitometry for imaging.
STP-8: TRANSPARENCY DRYING. All stained gels are air dried between cellophane sheets unless
otherwise requested. Although this treatment adds a day to turnaround, the
advantages of easier storage, little or no curling, good color retention,
overlay capability, scanning capability, and overhead projection capability
offset the time delay. Since cellophane quenches 35S and 3H,
gels with these isotopes scheduled for autoradiography or fluorography are paper
dried (STP-9). STP-9: PAPER DRYING This is an optional drying method for stained gels. All Enhance-treated slab gels scheduled for fluorography as well as unstained gels scheduled for autoradiography are dried onto thick filter paper unless otherwise requested. Price: No charge. STP-10: WET GELS
In some cases the return of
wet gels is requested, for example, for a core facility's automatic spot
picker. This is no problem. The wet gels are placed between sheets of
thick wet filter paper and the sandwich placed in a ziplock bag. The bag
is supported by pressboard sheets and cushioned by bubble wrap prior to
express mailing. Gel identification information is written on
the ziplock bag. 2D Gels/Top Comparisons Mass Spectrometry Staining Blotting Sample Preparation Misc RADIOACTIVITY PROCEDURESRP-1: AUTORADIOGRAPHY.
Includes 2 film exposures at room temperature using 8" x 10" Kodak BioMax MR
film. Exposure times are usually 2 and 4 days. RP-2: ENHANCE TREATMENT. For
fluorography of low energy emitters such as 3H, 14C, and 35S. (New England
Nuclear reagent and protocol). The gel is soaked in a solution (Enhance)
containing a fluorescent intermediate which, at low temperatures, converts
beta-particle energy into photons. This treatment reduces exposure times for
14C and 35S to about 1/8 that of untreated gels. Note that disposal costs
for used Enhance add to the cost. RP-3: FLUOROGRAPHY. Requires
prior Enhance treatment (RP-2). Includes 2 film exposures at -70oC using gel
8" x 10" Kodak BioMax MR film. Exposure times are usually 2 and 4 days. RP-4: 14C-MOLECULAR WEIGHT MARKERS.
These markers from Amersham are added during slab gel electrophoresis so that
bands appear on X-ray film after exposure of the gels. These markers have molecular weights of 200,000, 97,400, 69,000, 46,000, 30,000 and 14,300.
RP-6 RADIONUCLIDE DISPOSAL FEE
to help cover the high cost of our Radioactive Materials License and disposal fees. 2D Gels/Top Comparisons Mass Spectrometry Staining Radioactivity Sample Preparation MiscBLOTTING & WESTERN BLOTTING P-Tyr WB package P-Ser/P-Thr WB packageBP-1: TRANSBLOTTING ONTO PVDF. (Immobilon
from Millipore Corp.) PVDF binds proteins more tightly than nitrocellulose
and gives better recoveries. Transblotting onto PVDF is carried out
overnight according to standard procedures using a tris/glycine/methanol
buffer system without SDS. The dried stained (see BP-2) or unstained blot is
returned to the client. Staining facilitates matching to a duplicate
Coomassie gel for mass spectrometry. Original gels are stained to make sure
they’re blank and discarded, unless otherwise requested. We will change the
blotting procedure to your specifications if necessary. BP-2: PVDF COOMASSIE STAINING.
The 2D gel pattern obtained from Coomassie blue staining of a
PVDF membrane is faded but the scanned image (2D-ES-14, Electronic photos,
$15) can be contrasted with Adobe photoshop. The image is exactly
superimposable with the ECL film from Western blotting and helps to match a
few spots on the film to a busy 2D gel pattern on a duplicate Coomassie gel
run for mass spectrometry. Since Western blotting can be 100 times more
sensitive than Coomassie, careful matching is important. Coomassie staining
of the blot does not interfere with subsequent Western blotting. BP-3: TRANSBLOTTING ONTO NITROCELLULOSE.
Transblotting is carried out overnight according to standard
gel procedures using a tris/glycine/methanol buffer system containing 0.1%
SDS, with nitrocellulose of 0.45 micron pore size. Dried unstained blots are
returned to the client and original gels are discarded unless additional
treatments are requested. We will change the blotting procedure to your
specifications if necessary. BP-4: INDIA INK STAINING OF BLOTS.
Nitrocellulose cannot be Coomassie blue-stained or
silver-stained. India ink is the preferred method to visualize patterns on
nitrocellulose blots for purposes of comparisons with immuno-stained blots.
The method of Hancock and Tsang is used. (Anal Biochem. 133: 157, 1983). BP-5: COLORED MOLECULAR WEIGHT MARKERS FOR BLOTS.
These markers from Diversified Biotech are added during slab gel
electrophoresis; so that colored bands are visible on both unstained gels
and subsequent transblots at molecular weights of 95,500, 55,000, 43,000,
36,000, 29,000, 18,400, and 12,400. BP-6: WESTERN (IMMUNO-) BLOTTING, CLIENT SUPPLIES
ANTIBODY. This method for immuno-detection
of specific bands or spots is carried out on PVDF transblots from 1-D or 2-D
gels (see: "Handbook of Immunoblotting of Proteins Vol 1 and 11", Eds.
Bjerrum, O. and Heegaar, N., 1988, CRC Press, Inc; and "Gel Electrophoresis:
Proteins" by Dunn, M.J., 1993, Bios Scientific Publishers, Ltd). Requires
prior electrophoresis (2D-ES-1 or 1D-ES-1) but PVDF transblotting (BP-1) is
included in the fee. Generally the client supplies the antibody. The
sensitive ECL method of detection is used. (Amersham reagents and protocol
are used.) A control membrane run with secondary ab is included as a control
if necessary. BP-7:
INTRODUCTORY OFFER 40% OFF:
PHOSPHOTYROSINE WESTERN BLOTTING PACKAGE INCLUDING ANTIBODY. We have
optimized a method for
identifying phosphotyrosine-containing proteins by 2D Western blotting
using the PY20 anti-PTyr antibody. Our results suggest that the method is
specific, reproducible, and sensitive. However, in some cases the phosphate
bond is labile so duplicate Western blots are encouraged to verify
reproducibility. BP-8:
INTRODUCTORY OFFER 40% OFF: COMBINED P-SERINE/P-THREONINE WESTERN
BLOTTING PACKAGE USING ECL ADVANCE AND QIAGEN’S Q5 AND Q7 ABS.
We have optimized a method for 2D
Western blotting against phosphoserine and phospho-threonine residues on
proteins using the Q5 and Q7 antibodies from Qiagen and ECL Advance from GE
Healthcare. Both antibodies are advertised as detecting phosphorylated
residues irrespective of surrounding amino acids. The ultra sensitive ECL
Advance enables us to dilute the antibodies to 1:4000 and combine them for
this package. Duplicate Western blots are encouraged to verify
reproducibility. BP-9: INTRODUCTORY OFFER 40% OFF:
PHOSPHOSERINE OR PHOSPHOTHREONINE WESTERN BLOTTING.
Same package as above except single antibodies are used instead of a
mixture. 2D Gels/Top Comparisons Mass Spectrometry Staining Blotting Radioactivity MiscSAMPLE PREPARATION PROCEDURESSP-1: PROTEIN DETERMINATION
followed by buffer addition.
The total amount of protein in each sample is determined by the
Pierce BCA total protein assay. If you require protein determinations, do not
dissolve samples in buffers containing dithiothreitol or ßME. We will add
ßME after protein analysis. SP-2 SPECIFIC ACTIVITY (PROTEIN-BOUND DPM) DETERMINATION
This
measurement includes TCA precipitation, washing the pellet and then
determining cpm per mg protein. The method is described in our Sample
Preparation Guide. High concentrations of SDS (> 1.0%) add
variability to this method.
SP-3: ETHANOL PRECIPITATION OF PROTEINS
followed by buffer addition.
SP-4: NUCLEASE TREATMENT includes homogenization
with osmotic lysis buffer containing protease inhibitors followed by incubation
on ice with DNase and RNase to break down interfering polynucleotides. The
composition of the 10X Nuclease Stock Solution is given in our Sample
Preparation Guide. If you require nuclease treatment send the samples in a
buffer containing less than 0.3% SDS. Higher SDS concentrations inactivate
nucleases. SP-5: MICRODIALYSIS
followed by lyophilization & buffer addition. High salt concentration
(> 150 mM) interferes with IEF; the less salt the better. If your samples contain high salt or buffer
(Phosphates, NaCl, etc.), this dialysis and concentration step is highly recommended.
SP-6: LYOPHILIZATION
followed by buffer addition.
SP-7: TCA PRECIPITATION
followed by buffer
addition. SP-8: TCA/ACETONE PRECIPITATION follwed by buffer
addition. SP-9: CUSTOM SAMPLE PREP.
(Chunks of tissue, leaves, etc.) hr.
SP-10: MAJOR PROTEIN REMOVAL FROM HUMAN
SERUM, PLASMA & CSF. Mixed IgY affinity microbead columns from GenWay
www.genwaybio.com are used to remove
the 12 most abundant proteins from plasma or serum samples (albumin, IgG,
Fibrinogen, Transferrin, IgA, IgM, Haptoglobin, A2-Macroglobulin, A1-Acid
Glycoprotein, A1-Antitrypsin and HDL (Apo A-I & Apo-A-II). Genway “Supermix”
- inquire. 2D Gels/Top Comparisons Mass Spectrometry Staining Blotting Radioactivity Sample Preparation cGMP-1 CURRENT GOOD
MANUFACTURING PRACTICE The numerous SOPs required for
cGMP for 1D and 2D electrophoresis are in place at Kendrick Labs along with an
archival system for equipment calibrations and reports. However, additional
validations and archiving are required for new projects to pass detailed FDA
inspections - which may occur years down the road. Please let us know if you are
going to submit an IND, or plan to use our lab for quality assurance. CON-1 CONSULTING FEES Price: $135/hr. STORAGE, FREEZER LONG TERM:
Kendrick Labs will store your samples 1 year in our –80° C freezer without
charge. However, because of space constraints, we must now charge $150/6
months or $300/year for longer storage. 1D-FISH ANALYSIS: Fish Species
determination by the method of Pineiro, C., Barros-Velazquez, R.
Perez-Martin, I. Martinez, T. Jacobsen, H. Rehbein, R. Kundiger, R. Mendes,
M. Etienne, M. Jerome, A. Craig, I. Mackie and F. Jessen, “Development of a
SDS-polyacrylamide gel electrophoresis reference method for the analysis and
identification of fish species in raw and heat-processed samples: A
collaborative study”. Electrophoresis, 20: p1425-32 1999. Terms and Conditions |
