Western in A Day

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Western in A Day

• Ning Tingting, Ph.D
• Field Application Specialist, BIO-RAD
• Saturday, July 12, 2014

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Northern Blot
RNA
Western Blot Far Western Blot Dot Blot
?
Protein
DNA
Southern Blot
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Transfer Methods
The principle of Electrophoretic transfer
Proteins migrate to the membrane following a
current (I) that is generated by applying a
voltage (V) across the electrodes, following
Ohms law V I x R
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Transfer Methods
The principle of Electrophoretic transfer

• the applied voltage and the distance between the
  electrodes play a major role in governing the
  rate of elution of the proteins from the gel.
• the size, shape, and charge of the protein and
  the pH, viscosity, and ionic strength of the
  transfer buffer and gel T influence the elution
  of particular proteins from gels
• There are practical limits on field strength,
  however, due to the production of heat during
  transfer.

Joule heating is proportional to P I x V I2 x
R
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Transfer Methods
The principle of Electrophoretic transfer
Joule heating is proportional to P I x V I2 x
R
Joule heating ?

• Buffer resistance ?
• Buffer buffering capacity ?
• Inconsistent field strength and transfer
• Gel to deteriorate and stick to the membrane

The major limitation of any electrophoretic
transfer method is the ability of the chamber to
dissipate heat
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Transfer Methods
Types of Electrophoretic transfer
Tank Blotting Semi-Dry Blotting
Flexibility Flexible voltage settings, blotting times, and cooling requirements flexible electrode positions (Trans-Blot and Trans-Blot Plus cells) (Trans-Blot and Trans-Blot Plus cells) without cooling
Quantitative vs qualitative results Quantitative transfer of low molecular weight proteins possible under conditions that allow efficient binding to the membrane Some low molecular weight molecules will be transferred through the membrane without binding quantitatively
Molecular weight range Broad molecular weight range Variable transfer efficiencies for proteins gt120 kD (may be improved with discontinuous buffer system) low molecular weight proteins may be transferred through membrane
Transfer time Extended transfer (up to 24 hr) possible without buffer depletion rapid transfers (1560 min) obtained under high-intensity conditions Rapid transfers extended transfers not possible due to buffer depletion
Temperature control Specific temperature regulation with cooling coil and refrigerated water recirculator permits transfers at low temperatures (410C), for example, native enzyme transfers Temperature regulation by external cooling is not possible
Buffer capacity Up to 1012 L (Trans-Blot Plus cell) or as little as 450 ml (Mini Trans-Blot cell) length of blotting time not restricted by limited buffer capacity Minimal, 250 ml per experiment reduced cost of reagents and experiment time
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Membrane, Buffer Power conditions
Membrane selection
! PVDF membrane must be wetted in 100 methanol
prior to use but may be used with a transfer
buffer that contains no methanol
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Membrane, Buffer Power conditions
Transfer buffer selection
General recommandation

• SDS in buffer protein mobility
• âbinding efficiency to membrane
• SDS confers a negative charge to positive and
  neutral proteins
• increases transfer efficiency of large proteins
• recommended when using SDS PVDF or positively
  charged nylon membranes
• will increase the relative current, power and
  heating
• may affect the antigenicity of some proteins
• à discontinuous system with only 0.01 SDS

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Membrane, Buffer Power conditions
Transfer buffer selection
General recommandation

• Methanol in buffer
• â protein transfer from gel
• binding to nitrocellulose
• not necessary with PVDF or nylon membranes
• gel shrinks, the pores of the gel are reduced
• removes SDS from SDS-protein complexes
• precipitation, denaturing, loss of biologic
  activity

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Membrane, Buffer Power conditions
Transfer buffer selection
Standard Towbin buffer contains 25 mM Tris, pH
8.3, 192 mM glycine, 20 (v/v) methanol and,
occasionally, 0.0250.1 (w/v) SDS.
A buffer similar in composition to the standard
Towbin buffer is the Bjerrum and Schafer-Nielsen
buffer (48 mM Tris, pH 9.2, 39 mM glycine, 20
methanol), which was developed for use in
semi-dry applications
CAPS buffers (10 mM CAPS, pH 11, 10 methanol)
may be preferable for transfers of high molecular
weight proteins (for example, gt150 kD) and in
cases where the glycine component of Towbin
buffer may interfere with downstream protein
sequencing applications
Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3,
pH 9.9, 20 methanol) may produce higher
efficiency transfers and improve the ability of
antibodies to recognize and bind to proteins
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Membrane, Buffer Power conditions
Transfer buffer selection
Discontinuous Tris-CAPS Buffer System (Semi-Dry
Transfers)
60 mM Tris, 40 mM CAPS, pH 9.6, plus 0.1 SDS
60 mM Tris, 40 mM CAPS, pH 9.6, plus 15 methanol
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Membrane, Buffer Power conditions
Power conditions for Electrophoretic Transfers
Selecting power supply settings
During tansfer, the resistance decreases as a
result of Joule heating, except for semi dry
blotting where R increases due to ion depletion.
Transfer under constant voltage U cst R?
x I ?

• need a cooling system
• field strength will remain constant, providing
  the most efficient transfer possible

Transfer under constant current U? R? x I
cst

• heating minimized
• proteins will be transferred more slowly due to
  decreased field strength

Transfer under constant power P cst R ?
x I2 ? U2 ? / R ?

• current increases but less than for Vcst (v),
  decreased field strength
• alternative to constant current for regulating
  heat production during transfer

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Performing the transfer
Tips

• Remove all air bubbles at each step with a roller
• To avoid ghost prints and other artifacts, do not
  move the membrane and/or gel after it is
  positioned
• Place the transfer tank onto a magnetic stirplate
• The tanks are effective thermal insulators and
  limit the efficient dissipation of heat.
  Therefore, placing blotting cells in a coldroom
  is not an adequate means of controlling transfer
  buffer temperature. Use the internal cooling
  devices
• For SD, use one extra thick filter paper instead
  of two or three thin or thick filter paper to
  avoid air bubbles between them
• For SD, the membrane and filter paper should be
  cut to the same size as the gel

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Detection
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Detection
Detection Methods
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Detection
Immunological Detection Procedure
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Detection
Immunological detection methods
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What is important to a western blot experiment?

• Can I see my target proteins?
• if not, whats wrong?
• Transfer
• Sample
• Detection sensitivity
• if yes, how reliable?
• Accurate?
• Reproducible?
• Can I get away from the tedious work?

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Bio-Rad V3 WB workflow to address these issues

• Can I see my target proteins?
• if not, whats wrong?
• Transfer (Verify better monitoring of transfer
  efficiency)
• Sample (Visualize sample quality control)
• Detection sensitivity (more sensitive detection
  reagents)
• if yes, how reliable?
• Accurate? (Validate Quantification and loading
  control)
• Reproducible? (transparency of the whole process
  for better monitoring, such as loading error,
  uneven transfer etc.)
• Can I get away from the tedious work?
• Fast procedure
• Multiplexing

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Content

• What is Bio-Rad V3 Western Blot Workflow
• What is important to a western blot experiment?
• Visualize gel run
• Verify Transfer efficiency
• Validate quantitative analysis
• Multiplexing of the detection
• Comparison between Bio-Rad Bio-Rad V3 Western
  Blot Workflow and the traditional western blot
  approach

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What is it?

• Bio-Rad V3 Western Blot workflow is a Bio-Rad
  complete western blot solution incorporated with
  new technologies and products, such as TGX stain
  free gels, Trans-blot Turbo and ChemiDoc MP
  imager system, to provide a best practice in
  western blot experiment to ensure higher
  confidence in the experimental findings over the
  traditional western blot workflow.

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The traditional western blot workflow
Traditional WB Approach
Gel prep
By eye
By ChemiDoc MP (optional view under UV)
gt 1 h
Electrophoresis
1 h
blotting
1-3 h
Immunodetection
5 h
Imaging data analysis
gt30min
Often need reprobing 5h
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Visualize

• ????

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Bio-Rad Stain Free Technology
ChemiDoc MP system
Stain Free Pre-cast Gels
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Stain Free Technology

• 1. Stain-free compound is premixed with the gel,
  therefore no staining step required
• 2. Gel image can be generated 5 minutes after
  electrophoresis is finished
• 3. UV induced fluorescent gel or blot image
• 4. Low background produced and staining is
  uniform across the gel and blot
• 5. Non-reversible staining on both the gel and
  blot (can not wash away)
• 6. The same gel is used for both total protein
  loading control and transfer for western blotting

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Sample quality control Stain Free Technology
to Visualize the gels within 5 min after gel run
A total protein gel image allows a double check
of the protein profiles in each sample to avoid
artifacts caused by inaccurate protein assay
and/or protein degradations. The sample quality
control tells you when to terminate your
experiment Dont waste time and money on bad
samples
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Verify

• ????

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Fast Blotting of TGX Stain Free gels make it
easy to verify transfer efficiency
3 Min

• Trans-Blot Turbo with TGX gels

15 Min
Tank Blotting with TGX gels
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How do I know transfer was successful and how to
optimize it?

• Traditional approach
• only indicative, prestained protein standards
• no image recording, not quantitatively
• Bio-Rad V3 WB approach
• quantitative Stain Free Gel image
• true visualization of the protein samples
• To optimize transfer conditions
• easy and fast
• If some proteins remained in the gel after
  transfer, set up another blotting with the same
  gel to give another 5 min of transfer
• Add this extra time to the original setting for
  next blotting experiment

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Now you can see proteins on the gels before
transfer
Stain free image of protein samples on the Gel
Double check your loading consistency Double
check the sample quality
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Now you can see proteins on the post-transfer gels
Stain free image of a Post-Blot Gel
No more guess about the transfer efficiency. Now
you have the control you can calculate how many
percent of the total protein are still left in
the post-transfer gel
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Now you can see proteins on the blot
Stain Free image of proteins on a blot
Worry about protein loss during striping for
reprobing? No more worries, now you have the
control simply put the blot in the ChemiDoc MP
to take a quick look
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Validation????

• Linear dynamic ranges
• Loading control
• Western Blot can be more quantitative

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Introduction to Western Blot Validation

• Validation of western blot results Why and How?
• How? Typically, a specific housekeeping protein
  is used as loading control (GAPDH, Actin,
  Tubulin)
• Experimental errors are identified by
  quantitative differences in HKP
• Target protein data is normalized to the HKP
  signal
• Most customers do chemiluminescence detection ?
  Strip and reprobe or cut the membrane
• Sounds easy, right? Whats the problem with both
  experiments below?

Experiment A
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What is loading control?
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What are the commonly used loading control
proteins?
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Samples of loading control in publication -- PNAS
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Whats wrong?
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Whats wrong?
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Whats wrong?

• Western Blotting Help!!!! - Problem with an
  internal control (Jul/28/2004 )
• Hello ALL!Im hoping someone can explain what
  is going on with some of my data!!!!We are
  currently checking cell viability post cell
  explant from saphenous vein tissue. Our viability
  data indicates that we are isolating less cells
  from the tissue as time increases and that these
  cells in culture are less viable. So to look at
  specific cell markers we thought it locgical to
  run our samples on a SDS-PAGE and use Beta Actin
  as a loading control. Time after time we are
  loading equal amounts of protein per lane but our
  cell specific marker is decreasing (which is
  logical...that is the amount of cells expressing
  H-caldesmon.....) but our B-actin which should be
  ubiquitous to all cells is also decreasing at a
  similar ratio. This doesnt make sense to me as we
  were hoping to see loss of protein cell-specific
  protein, only!! We have used BCA to measure our
  protein concentations in our cell lysates, and
  have used kits from different manufacturers with
  similar results. We have used different
  antibodies, both monoclonal and polclonal. We
  have also used different H-caldesmon antibodies.
  Each time with similar results. Could cell death
  (which we know by our viability experiments)
  contribute to the decrease in beta-actin (our
  internal control) despite the loading of equal
  amounts of protein. By the way, this has also
  been done with GAPDH and tubulin. I'm at a loss
  and cant explain this.PLEASE HELP?!!!

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Doubt

• ELECTROPHORESIS Volume 27, Issue 14, pages
  28442845, No. 14 July 2006
• ß-Actin is not a reliable loading control in
  Western blot analysis
• Angela Dittmer, Jürgen Dittmer Dr.
• Abstract
• ß-Actin is often used as a loading control in
  Western blot analysis. We analyzed the ability of
  ß-actin-specific antibodies to recognize
  differences in protein loading. We found that, at
  higher total protein loads as required for the
  detection of low-abundance proteins,
  ß-actin-specific antibodies failed to distinguish
  differences in actin protein levels. Diluting the
  antibody working solution or changing the
  incubation time had little effect on this
  phenomenon. This shows that ß-Actin is not a
  reliable loading control in Western blot
  analysis. In general, it appeared that, at longer
  incubation times, antibodies seem to be less able
  to pick up differences in the level of its target
  protein.

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Doubt
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ß-actin varies in some situations

• Varying expression level of ß-actin in
  response to biomedical stimuli
• Altered expression of ß-actin during growth
  and differentiation
• Changed expression of ß-actin in some diseases

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ß-actin varies in some situations
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Expression difference _at_PCR level
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Reason 2 overexposure
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In the case of the polyclonal anti-b-actin
antibody, no change in band intensity could
be observed in the range of 7.51.88 mg of total
protein. At 0.94 mg protein the signal gradually
decreased and was undectable at 0.12 mg or lower.
The monoclonal anti-b-actin antibody generated a
similar picture, except that similar band
intensities were found in a broader range of
protein loads between 0.47 and 7.5 mg.
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What are the alternatives for loading control?
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What are the alternatives for loading control?
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What are the alternatives for loading control?
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Option 1- Irreversible

• Coomassie staining
• Silver staining
• Amido black staining
• Fluoresce staining

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Option 2 - reversible

• Analytical Biochemistry Volume 401, Issue 2, 15
  June 2010, Pages 318-320
• Reversible Ponceau staining as a loading control
  alternative to actin in Western blots
• Isabel Romero-Calvoa, Borja Ocóna, Patricia
  Martínez-Moyaa, María Dolores Suáreza, Antonio
  Zarzueloa, Olga Martínez-Augustina and Fermín
  Sánchez de Medina , a, a
• Departments of Biochemistry and Molecular
  Biology II and Pharmacology, School of Pharmacy,
  University of Granada, Centro de Investigación
  Biomédica en Red de Enfermedades Hepáticas y
  Digestivas (CIBERehd), Campus de Cartuja s/n,
  18071 Granada, Spain
• Abstract
• It is becoming standard practice to measure a
  housekeeping gene, typically actin, in Western
  blots, as it is the rule in RNA blots. We have
  applied reversible Ponceau staining to check
  equal loading of gels and measured actin in
  parallel under different conditions. Our results
  show that densitometric analysis is comparable
  with both techniques. Therefore, routine
  quantitation of Ponceau staining before antibody
  probing is validated as an alternative to actin
  blotting.
• Loss of target, Waste of membrane and time

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How to measure total protein local on a stain
free image

1. Acquire a stain free gel image
2. Using the Image Lab software Volume Tools to
   define a volume area to cover the whole lane of a
   sample
3. Copy and paste the defined the area to cover
   other sample lanes
4. The total protein amount is measured by the Adj.
   Vol. in the data analysis table

1 2 3 4 5 6 7
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Bio-Rad solution to a more reliable loading
control
TGX Stain Free Precast Gels ChemiDoc MP Imager
System provide The best way to measure total
proteins for loading controls in a western blot
experiment
Stain Free Pre-cast Gels
ChemiDoc MP system
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Benefits of the Bio-Rad V3 WB Workflow over
traditional approaches
Traditional WB Approach
Faster and More Reliable WB workflow
Gel prep
Precast TGX stain free gel
gt 1 h
Electrophoresis
15-20 min
1 h
blotting
1-3 h
Turbo
3-15 min
Immunodetection
5 h
5 h
Imaging data analysis
gt30min
Often need reprobing 5h
No need to reprobe
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Multiplexing WB to analyze phosphoproteins
Nature Methods 2, 79 - 81 (2005) Western blot
analysis with quantum dot fluorescence
technology a sensitive and quantitative method
for multiplexed proteomics
Western blot images of p42 MAPK (a, green) and
phosphorylated p42 MAPK (b, red) expression in
serum-starved 3T3 cells in response to PDGF
stimulation. Cells were harvested at 0, 10, 20,
40 and 60 min after PDGF administration (lanes
15, respectively). The anti-pan MAPK primary
antibody (a) was detected with Qdot 605 nm
Conjugate, a red-orange dot that was
pseudocolored green and the phospho-MAPK primary
antibody (b) was detected with Qdot 705 nm
Conjugate and pseudocolored red. (c) Overlay of
images in a and b.
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Multiplexing WB to detect 3 different targets on
the same blot using Qdot Conjugates
Excitation 415nm /100nm filter Green Anti-GST
, 565nm Red Anti-GST-HA, (rabbit) ,
605nm, Blue Anti-GST-cMyc (mouse) 705nm
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Multiplexing WB to detect 4 different targets on
the same blot
Hsc70 (green), beta-actin (red) and ERX1 and ERX2
(blue)
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Titration of lac operator DNA with lac repressor
protein. Increasing amounts of lac repressor
protein were mixed with 40 ng of lac operator
DNA, incubated for 20 minutes and then separated
on a 6 nondenaturing PAGE. The gel was stained
with SYBR Green EMSA stain (green) followed by
SYPRO Ruby EMSA stain (red), components of the
Electrophoretic Mobility-Shift Assay Kit . After
each staining, the image was documented using an
FLA-3000 laser-based scanner (Fuji) and the
digital images pseudocolored and overlaid. Yellow
bands indicate areas stained with both stains.
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This fluorescent western blot shows simultaneous
detection of unphosphorylated and phosphorylated
Akt1 present in serum starved and PDGF stimulated
NIH/3T3 whole cell lysates. Lane 1, unstimulated
NIH/3T3 lysates contain inactive unphosphorylated
Akt1, green band. Lane 2, PDGF stimulated NIH/3T3
lysate contains both inactive (green band) and
activated phosphorylated Akt1 (red band). Both
lanes were probed with rabbit anti-Akt (pan) and
mouse anti-Akt pS473 specific antibodies followed
by detection with DyLight 549 conjugated
anti-rabbit IgG (green) and DyLight 649
conjugated anti-mouse IgG (red) secondary
antibodies.
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The Bio-Rad V3 WB workflow

• Can I see my target proteins?
• if not, whats wrong?
• Better monitoring of transfer efficiency stain
  free gels and ChemiDoc MP imager
• Sample quality control stain free gels and
  ChemiDoc MP imager
• More sensitive detection reagents ChemiDoc MP
  imager and WesternC Chemiluminescence kit
• if yes, how reliable?
• Loading control stain free gels and ChemiDoc MP
  imager
• transparency of the whole process for better
  monitoring ChemiDoc MP imager, stain free gels
  and low fluorescence PVDF membrane
• Can I get away from the tedious work? (fast)
• TGX gels and Trans Blot Turbo
• Fluorescent multiplexing on ChemiDoc MP imager

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3806255-805 ????18627760323