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Mouse Leptin（ （ （ （LEP） ） ） ）ELISA kit
Catalog No. CSB-E04650m
（96T）
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of mouse
LEP  concentrations  in  cell  culture  supernates,  serum,  plasma  and  other
biological fluids.
  Expiration date      six months from the date of manufacture
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 
 
 
 
 
CUSABIO BIOTECH CO., Ltd.
/    /
: cusabio@    cusabio@ 
  2
INTRODUCTION
Leptin  （from  the  Greek  leptos, meaning  thin）  is  a  protein  hormone  with
important  effects  in  regulating  body weight, metabolism  and  reproductive
function. The protein is approximay ~16 kDa in mass and encoded by the
obese  （ob） gene. Leptin  is expressed predominantly by adipocytes, which
fits with the idea that body weight is sensed as the total mass of fat in the
body. Smaller amounts of leptin are also secreted by cells in the epithelium
of the stomach and in the placenta. Leptin receptors are highly expressed in
areas of the hypothalamus known to be important in regulating body weight,
as well as in T lymphocytes and vascular endothelial cells.  
PRINCIPLE OF THE ASSAY
The  microtiter  plate  provided  in  this  kit  has  been  pre-coated  with  an
antibody  specific  to  LEP.  Standards  or  samples  are  then  added  to  the
appropriate  microtiter  plate  wells  with  a  biotin-conjugated  polyclonal
antibody preparation  
specific for LEP. and Avidin conjugated to Horseradish Peroxidase （HRP） is
added  to  each  microplate  well  and  incubated.  Then  a  TMB  （3,3'5,  5'
tetramethyl-benzidine） substrate solution is added to each well. Only those
wells that contain LEP., biotin-conjugated antibody and enzyme-conjugated
Avidin  will  exhibit  a  change  in  color.  The  enzyme-substrate  reaction  is
terminated by the addition of a sulphuric acid solution and the color change
is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The
concentration of LEP. in the samples is then determined by comparing the
O.D. of the samples to the standard curve. 
  3
DETECTION RANGE
0.16  ng/ml-10  ng/ml.  The  standard  curve  concentrations  used  for  the
ELISA’s were  10  ng/ml,  5  ng/ml,  2.5  ng/ml,  1.25  ng/ml,  0.62  ng/ml,  0.32
ng/ml, 0.16 ng/ml
SPECIFICITY
This assay recognizes recombinant and natural mouse LEP No significant
cross-reactivity or interference was observed.
SENSITIVITY
The minimum  detectable  dose  of mouse  LEP  is  typically  less  than  0.04
ng/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined
as the lowest protein concentration that could be differentiated from zero.
MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standard  2
Sample Diluent      1 x 20 ml
Biotin-antibody Diluent      1 x 10 ml
HRP-avidin Diluent        1 x 10 ml
Biotin-antibody      1 x 120µl
HRP-avidin  1 x 120µl
Wash Buffer      
1 x 20 ml
  （25×concentrate）
TMB Substrate      1 x 10 ml
Stop Solution      1 x 10 ml 
  4
STORAGE
1.  Unopened  test  kits  should  be  stored  at  2-8°C  upon  receipt  and  the
microtiter  plate  should  be  kept  in  a  sealed  bag  with  desiccants  to
minimize exposure to damp air. The test kit may be used throughout the
expiration date of  the kit. Refer  to  the package  label  for  the expiration
date.
2.  Opened  test  kits  will  remain  stable  until  the  expiring  date  shown,
provided it is stored as prescribed above.    
3.  A  microtiter  plate  reader  with  a  bandwidth  of  10  nm  or  less  and  an
optical  density  range  of  0-3  OD  or  greater  at  450nm  wavelength  is
acceptable for use in absorbance measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.  
1.  Wash Buffer    If crystals have  formed  in  the concentrate, warm up  to
room  temperature  and  mix  gently  until  the  crystals  have  compley
dissolved. Dilute 20 ml of Wash Buffer Concentrate  into deionized or
distilled water to prepare 500 ml of Wash Buffer.
2.  Standard    Reconstitute the Standard with 1.0 ml of Sample Diluent.
This  reconstitution  produces  a  stock  solution  of  10  ng/ml.  Allow  the
standard to sit for a minimum of 15 minutes with gentle agitation prior to
making  serial  dilutions.  The  undiluted  standard  serves  as  the  high
standard （10 ng/ml）. The Sample Diluent serves as the zero standard
（0 ng/ml）. 
  5
3.  Biotin-antibody    Dilute  to  the working concentration specified on  the
vial label using Biotin-antibody Diluent（1:100）, respectively.
4.  HRP-avidin    Dilute  to  the working concentration specified on  the vial
label using HRP-avidin Diluent（1:100）, respectively.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
  Microplate  reader  capable  of measuring  absorbance  at  450  nm, with
the correction wavelength set at 540 nm or 570 nm.
  Pipettes and pipette tips.
  Deionized or distilled water.
  Squirt bottle, manifold dispenser, or automated microplate washer.
SAMPLE COLLECTION AND STORAGE
  Cell Culture Supernates    Remove particulates by centrifugation and
assay immediay or aliquot and store samples at -20° C. Avoid
repeated freeze-thaw cycles.
  Serum    Use a serum separator  tube （SST） and allow samples  to clot
for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove
serum and assay  immediay or aliquot and store samples at  -20° C.
Avoid repeated freeze-thaw cycles.
  Plasma    Collect plasma using citrate, EDTA, or heparin as an
anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes
of collection. Assay immediay or aliquot and store samples at -20° C.
Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay. 
  6
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
1.  Add  100µl  of  Standard,  Blank,  or  Sample  per  well.  Cover  with  the
adhesive strip. Incubate for 2 hours at 37° C.  
2.  Remove the liquid of each well, don’t wash.  
3.  Add 100µl of Biotin-antibody working solution  to each well.  Incubate
for  1  hour  at  37°C.  Biotin-antibody  working  solution  may  appear
cloudy. Warm  up  to  room  temperature  and  mix  gently  until  solution
appears uniform.
4.  Aspirate each well and wash,  repeating  the process  three  times  for a
total of three washes. Wash by filling each well with Wash Buffer （200µl）
using  a  squirt  bottle,  multi-channel  pipette,  manifold  dispenser  or
autowasher.  Complete  removal  of  liquid  at  each  step  is  essential  to
good  performance. After  the  last wash,  remove  any  remaining Wash
Buffer  by  aspirating  or  decanting.  Invert  the  plate  and  blot  it  against
clean paper towels.
5.  Add  100µl  of  HRP-avidin  working  solution  to  each  well.  Cover  the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37° C.
6.  Repeat the aspiration and wash three times as step 4.
7.  Add 90µl of TMB Substrate  to each well.  Incubate  for 30 minutes at
37°C.  Keeping  the  plate  away  from  drafts  and  other  temperature
fluctuations in the dark.
8.  Add  50µl  of  Stop  Solution  to  each  well.  If  color  change  does  not
appear uniform, gently tap the plate to ensure thorough mixing.
9.  Determine  the optical density of each well within 30 minutes, using a
microplate reader set to 450 nm. 
  7
CALCULATION OF RESULTS
Average the duplicate readings for each standard, control, and sample and
subtract the average zero standard optical density. Create a standard curve
by reducing the data using computer software capable of generating a four
parameter  logistic  （4-PL）curve-fit. As  an  alternative,  construct  a  standard
curve  by  plotting  the mean  absorbance  for  each  standard  on  the  y-axis
against the concentration on the x-axis and draw a best fit curve through the
points on  the graph. The data may be  linearized by plotting  the  log of  the
LEP. concentrations versus the log of the O.D. and the best fit line can be
determined  by  regression  analysis.  This  procedure  will  produce  an
adequate but less precise fit of the data. If samples have been diluted, the
concentration  read  from  the  standard  curve  must  be  multiplied  by  the
dilution factor.
LIMITATIONS OF THE PROCEDURE
  The kit should not be used beyond the expiration date on the kit label.
  Do not mix or substitute reagents with those from other lots or sources.
  It is important that the Calibrator Diluent selected for the standard curve
be consistent with the samples being assayed.
  If samples generate values higher than the highest standard, dilute the
samples with the appropriate Calibrator Diluent and repeat the assay.
  Any  variation  in  Standard  Diluent,  operator,  pipetting  technique,
washing  technique,  incubation  time  or  temperature,  and  kit  age  can
cause variation in binding.
  This assay  is designed  to eliminate  interference by soluble  receptors,
binding proteins, and other  factors present  in biological samples. Until
all  factors  have  been  tested  in  the  Quantikine  Immunoassay,  the
possibility of interference cannot be excluded. 
  8
TECHNICAL HINTS
  When mixing or reconstituting protein solutions, always avoid foaming.
  To avoid cross-contamination, change pipette tips between additions of
each standard  level, between sample additions, and between  reagent
additions. Also, use separate reservoirs for each reagent.
  When  using  an  automated  plate  washer,  adding  a  30  second  soak
period  following  the  addition  of wash  buffer,  and/or  rotating  the  plate
180 degrees between wash steps may improve assay precision.
  To  ensure  accurate  results,  proper  adhesion  of  plate  sealers  during
incubation steps is necessary.
  Substrate  Solution  should  remain  colorless  until  added  to  the  plate.
Keep Substrate Solution protected from light. Substrate Solution should
change from colorless to gradations of blue.
  Stop Solution  should be added  to  the plate  in  the  same order as  the
Substrate Solution. The color developed in the wells will turn from blue
to  yellow  upon  addition  of  the Stop Solution. Wells  that  are  green  in
color indicate that the Stop Solution has not mixed thoroughly with the
Substrate Solution. 
 
 

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