NovaTeinBio is a biological reagents provider located in the greater Boston area, USA.

Their scientific team is a group of scientists with extensive experience and expertise in protein biochemistry, proteomics, and immunoassay platforms.

They offer recombinant proteins, antibodies, ELISA kits and ELISA arrays.

Novatein believe that that if you succeed in your research, they succeed in their business.

That philosophy is at the centre of everything they do. It is why they work hard to deliver the highest-quality products and services on time and within budget. It is why they strive to provide all their customers with first-class service each and every day.


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Protocols

The following protocol is an outline of a traditio nal Western blotting protoc ol for the detection and characterization of a particular protein or bio-molecu le in a sample. Protein sample mixtures are first resolved by size using sodium dodecyl sulfate po lyacrylamide gel electrophoresis (SDS-PAGE). Then electro-transfer, or blotting of samples on the gel to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane is performed. Specific proteins are then detected on the blot by a series of blocking, incubating and washing steps with the appropriate buffers and antibody probes. Wester n blots resolve complex populations of proteins and provide data on the molecular weight and abundance of a particular protein of interest in a sample mixture.

Important NOTES:

  • A single-page quick protocol is included at th e end of this document. Print this page for quick bench-top reference.
  • Protein sample preparation methods vary widely in order to deliver protein in the conformation required for a particular assay. In this protocol, denaturing and reducing conditions are used as they are most common ly applied to Western blotting procedures and typically provide for the highest sensitivity and best resolution. However, users can make changes to the sample preparation and PAGE procedures in order to run non-denaturing or non-reducing conditions. Non-reducing conditio ns require no change to the protocol apart from the exclusion of reducing reagents from preparative buffers. Non-denaturing conditions require a more dramatic change in the PAGE protocol, and is not elucidated here.
  • Western blot probes vary widely. Antibodi es can be selected with a wide range of specificities and reactivities to a protein of in terest. This protocol details the generic methodology utilizing a primary antibody for protein recognition followed by a secondary antibody. The secondary antibody, which is against the primary, and conjugated to a chemiluminescence signal amplification enzyme, Horseradish Peroxidase (HRP) for visualization. Alternative probes may have othe r blotting requirements not included in this protocol. If so, consult with the manufacturer ’s guidelines of the reagent in question for further instructions.

Procedures

1.Prepare 2x LDS Buffer with 2x DTT:

Reagent Volume per Sample (μL) Sample Number Preparative Volume (μL)
4x LDS Sample Buffer 10 x _____ =
1M DTT 4 x _____ =
Deionized Water 6 x _____ =

 

 

 

2.Prepare protein samples for loading. Add 15uL of protein sample (diluted in deionized water if necessary) to 15uL of 2x LDS buffer containing 2x DTT.

3. Boil samples at 100 o C for 5 minutes.

4. Set up the PAGE gel. Quickly rinse the gel with deionized water, remove the comb and rinse the wells with running buffer, place it in the electrophoresis chamber, fill the upper trough with 1x running buffer.

5. Load 15uL of each prepared sample into a well on the gel in an established order.

6. Run the gel at a constant 180V for approximate ly 45-60 minutes or watch the position loading dye to stop the run.

7. Setup blotting sandwich and apparatus. Crack open the gel casing to access the gel. Cut away the wells and thick bottom portion of the gel. In a tray filled with 1x transfer buffer, assemble the blotting sandwich in the following order: (bottom) – filter-paper – gel – nitrocellulose – filter- paper – (top) . Place the blotting sandwich on the electroblotting apparatus as follows:

8. Block the membrane in 5% milk, 1x TBST fo r 30 minutes to 1hr on a rotating shaker.

9. While blocking, dilute the primary antibody in 5% milk, 1x TBST. Prepare enough diluted antibody to cover the blot during shaking incubation.

10. Decant blocking solution, rinse the membrane with 1x TBST, wash the membrane 2 – 3 times with 1x TBST buffer, each time 5 – 8 minutes with shaking on a rotating shaker.

11. Incubate the membrane with the diluted primary antibody for 30 minutes to 1hr on a rotating shaker.

12. If no secondary antibody or additional probes are necessary, proceed directly to step 10. Otherwise, discard the diluted primary antibody an d wash with 5% milk, 1x TBST for 5 – 8 mins on a rotating shaker. Repeat this wash step 2 additional times for a total of 3 washes.

13. While washing, dilute the secondary antibody in 5% milk, 1x TBST. Prepare enough diluted antibody to cover the blot during shaking incubation.

14. After washing, incubate the membrane with the diluted secondary antibody. Incubate for 30 minutes to 1hr on a rotating shaker.

15. Discard the diluted primary/secondary antibody and wash with 1x TBST for 3 times, each time 5 – 8 minutes on a rotating shaker.

16. Proceed with the appropriate ECL substrate incubation and chemiluminescence/fluorescence detection as specified by the reagents manufacturer.

Reagents & Buffer

1x SDS-PAGE Running Buffer (1L)Note: This buffer can be re-used multiple times. Replace buffer if its color changes or if gel-running is abnormal.50mL 20x NuPAGE MOPS SDS Running Buffer

950mL Deionized Water

1x Western Transfer Buffer (1L)Note: This buffer can be re-used multiple times. Replace buffer if its color changes or if gel transfer is abnormal.50mL 20x NuPAGE Transfer Buffer

200mL 99-100% Methanol

750mL Deionized Water

1x TBST Buffer (1L)100mL 10x Tris-Buffered Saline (500mM Tris pH 7.4, 1.5M NaCl)10mL 10% Tween-20

890mL Deionized Water

1x TBST Buffer + 5% Milk (1L)Note: Powdered milk must be thoroughly mixed into buffer before Tween addition so that no sediment remains.100mL 10x Tris-Buffered Saline (500mM Tris pH 7.4, 1.5M NaCl)

10mL 10% Tween-20

50g Blotting Grade Non-fat Dry Milk

890mL Deionized Water (Bring final volume to 1L)

Disposables/Reagents
PAGE Gel, 4-12%, or 4 -20 % Bis-Tris
SDS Sample Buffer (4x)
Western Blot Protein Standard
Dithiothreitol (DTT)
Beta-mercaptoethanol
MOPS or MES SDS Running Buffer (20x)
NuPAGE Transfer Buffer (20x)
Laboratory Grade Methanol
Blotting Sandwiches
Tris-Buffered Saline (10x)
Tween-20
Blotting Grade Non-fat Dry Milk
TBS Buffer
SuperSignal West Dura Extended Duration Substrate
Equipment
Gel box
Power Supply (100-120/220-240V)
Semi-dry Electroblotting System
Chemidoc Image System or X-ray film

The following protocol is a simplified alternative method , the Dot Blot, to traditional Western blotting for the detection of the presence or absence of a particular protein or bio-molecule in samples. Dot Blot differs from Westerns in that proteins in the samples are not resolved by size prior to blotting. This method is better suited for comparin g relative abundance of a target protein in a number of samples in parallel ( low-, medium-, high-throughput ).

Procedures

  • 1.Label the nitrocellulose blotting membrane for th e ease to locate your individual sample after blotting. For example, for 96-well sample dot bl ots, designate one corner of the membrane as well number A1, another corner as H12. Use indelible marker that will not bleed during processing.
  • 2.Place the labeled membrane on a filter paper. Se cure the edges of the membrane with tape or a weight to prevent the edge curling.
  • 3.Pipette 0.5-2.0uL of each sample onto separate pre-determined locations on the blot. A multi- channel pipette/96 or 384-pin head may be used to spot multiple samples at once. The samples should be absorbed to the membrane immediately without any beading on the surface.
  • 4.Block the membrane in 5% milk, 1x TBST fo r 30 minutes to 1hr on a rotating shaker.
  • 5.While blocking, dilute the primary antibody in 5% milk, 1x TBST. Prepare enough diluted antibody to cover the blot during shaking incubation.
  • 6.Decant blocking solution, rinse the membrane with 1x TBST, wash the membrane 2 – 3 times with 1x TBST buffer, each time 5 – 8 minutes with shaking on a rotating shaker.
  • 7.Incubate the membrane with the diluted primary antibody for 30 minutes to 1hr on a rotating shaker.
  • 8.If no secondary antibody or additional probes are necessary, proceed directly to step 10. Otherwise, discard the diluted primary antibody an d wash with 5% milk, 1x TBST for 5 – 8 mins on a rotating shaker. Repeat this wash step 2 additional times for a total of 3 washes.
  • 9.While washing, dilute the secondary antibody in 5% milk, 1x TBST. Prepare enough diluted antibody to cover the blot during shaking incubation.
  • 10.After washing, incubate the membrane with the diluted secondary antibody. Incubate for 30 minutes to 1hr on a rotating shaker.
  • 11.Discard the diluted primary/secondary antibody and wash with 1x TBST for 3 times, each time 5 – 8 minutes on a rotating shaker.
  • 12. Proceed with the appropriate ECL substrate incubation and chemiluminescence/fluorescence detection as specified by the reagents manufacturer. A sample Dot-Blot (spotted by 384- pin head ), with 0.5 ul/spot:

Reagents & Buffer

1x TBST Buffer (1L100mL 10x Tris-Buffered Saline (500mM Tris pH 7.4, 1.5M NaCl)

10mL 10% Tween 20

890mL Deionized Water

1x TBST Buffer + 5% Milk (1L)Note: Powdered non-fat dry milk must be thoroughly dissolved into buffer before Tween-20 addition so that no sediment remains.100mL 10x Tris-Buffered Saline (500mM Tris pH 7.4, 1.5M NaCl)

10mL 10% Tween 20

50g Blotting Grade Non-fat Dry Milk

890mL Deionized Water

Technical Information

Kit name and catalog number

Your analyte ELISA Kit, Catalog#: *****

Intended use

The kit is used to detect the level of Your analyte in cell culture, serum, blood plasma and other suitable sample solution.

Assay principle

The coated well immunoenzymatic assay for the quantitative measurement of analyte utilizes a multiclonal anti‐analyte antibody and an analyte‐HRP conjugate. The assay sample and buffer are incubated together with analyte‐HRP conjugate in pre‐coated plate for one hour. After the incubation period, the wells are decanted and washed five times. The wells are then incubated with a substrate for HRP enzyme. The product of the enzyme‐substrate reaction forms a blue colored complex. Finally, a stop solution is added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the analyte concentration since analyte from samples and analyte ‐HRP conjugate compete for the anti‐ analyte antibody binding site. Since the number of sites is limited, as more sites are occupied by analyanalytete from the sample, fewer sites are left to bind analyte‐HRP conjugate. Standards of known analyte concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color (Optical Density) to the concentration of analyte. The analyte concentration in each sample is interpolated from this standard curve.

Manufactured by:

NovaTein Biosciences 310 West Cummings Park Woburn, MA 01801 US

Telephone: (888) 856‐2858

 

Materialssupplied

1 Microelisa Stripplate 96 well 6 Chromogenic Substrate A 6ml X 1vial
2 Standard 1.0 ml X 6  vials 7 Chromogenic  Substrate B 6ml X 1 vial
3 100 X Wash Solution 10ml X 1vial 8 Stop Solution 6ml X 1 via
4 Lysis Buffer Solution 6ml X 1vial 9 Specification 1
5 5 HRP‐Conjugate Reagent 6ml X 1 vial

Note: Standard (S1 → S6) concentration was followed by:Variable with different kit

Sample collection and storage

  • Serum‐Use a serum separator tube (SST) and allow samples to clot for 30 minutes before a centrifugation for 15minutes at approximately 1000 x g. Remove serum and perform the assay immediately or aliquot and store samples at ‐20 °C or ‐80 °C.
  • Plasma‐Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2‐8 °C within 30minutes of collection. Store samples at  ‐20°C or ‐80°C.Avoid repeated freeze‐thaw cycles.
  • Cell culture fluid and other biological fluids‐Remove particulates by centrifugation and assay immediately or aliquot and store samples at  ‐20°C or  ‐80°C. Avoid repeated freeze‐thaw cycles.
  • NOTE: The Lysis Buffer Solution is used only when the sample is cell culture fluid & body fluid & tissue homogenate; if the sample is serum or blood plasma, then the Lysis Buffer Solution is a superfluous reagent. Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2‐8 °C, otherwise samples must be stored at  ‐20°C(≤2months) or ‐80°C(≤6months) to avoid loss of bioactivity and contamination. Avoid freeze‐thaw cycles. When performing the assay, warm up samples to room temperature slowly. DO NOT USE HEAT‐TREATED SAMPLES.

Materialsrequired but notsupplied

1.37 °C incubator

2.Standard plate reader capable of measuring absorbance at 450 nm.

3.Precision pipettes and disposable pipette tips

4.Distilled water

5.Multi‐channel pipettes, manifold dispenser or automated microplate washer.

6.Absorbent paper

Sample Preparation

1.Novateinbio is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient amount ofsamplesin advance.

2.Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.

3.If the samples are not indicated in the manual, a preliminary experiment to determine the validity ofthe kitis necessary.

4 Owing to the possibility of mismatching between antigen from other resource and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteinsfrom other manufacturers may not be recognized by our products.

5.Influenced by the factorsincluding cell viability, cell number and also sampling time,samplesfrom cell culture supernatant may not be detected by the kit

6.Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occurin those samples and finally lead to wrong results.

Reagent Preparation

1.Bring all kit components and samplesto room temperature (18‐25 ℃) before use.

2.Dispense 10 μl of Lysis Buffer Solution into 100μl specimens, mix and stand for one hour (The proportion of Lysis Buffer and Specimens should be no less than 1:10). (NOTE: This step is required when the sample is cell culture fluid & body fluid & tissue homogenate; if the sample is serum or blood plasma,then thisstep should be skipped.)

3. Wash Solution  ‐  Dilute 10 mL of Wash Solution concentrate (100×) with 990 mL of deionized or distilled water to prepare 1000 mL of Wash Solution (1×)

Assay procedures

Prepare all Standards before starting assay procedure (Please read Reagents Preparation). It is recommended that all Standards and Samples be added in duplicate to the Microtiter Plate.

1.Secure the desired numbers of coated wellsin the holder then add 100μl of Standards or Samples to the appropriate well of the antibody pre‐coated Microtiter Plate. Generally, you should get the sample value within the assay arrange without dilution. If samples generate values higher than the highest standard, further dilute the samples with 0.1%BSA in PBS, pH7.4 (without Mg2+, Ca2+) and repeat the assay.

2.Add 50μl of Conjugate to each well. Mix well.  Mixing well in this step is important. Cover and incubate the plate for 1 hour at 37 °C  .

3. Wash the Microtiter Plate using one of the specified methodsindicated below:   Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Fill in each well completely with diluted wash solution, and then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure five times for a total of FIVE washes. After washing, invert plate, and blot dry by hitting the plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that allstripsremain securely in frame. Complete removal of liquid at each step is essential to good performance. Automated Washing: Wash plate FIVE times with diluted wash solution (350‐400 μl/well/wash)   using an auto washer. After washing, dry the plate as above. It isrecommended that the washer be   setfor a soaking time of 10 seconds and shaking time of 5 seconds between each wash.

4.Add 50μl Chromogenic Substrate A and 50μl Chromogenic Substrate B to each well, subsequently. Cover and incubate for 10 minutes at 20‐25 °C . (Avoid sunlight).

5.Add 50μl of Stop Solution to each well. Mix well.

6.Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader immediately.

Important notes

1.The operation should be carried outin strict accordance with the provided instructions.

2.To preserve unused strip‐wells, itshould be stored in the sealed bag.

3.Always avoid foaming whenmixing orreconstituting protein solutions.

4.Pipette reagents and samplesinto the center of eachwell.

5.The samples should be transferred into the assay wells within 15 minutes of dilution.

6.We recommended that all standard, testing samples are tested in duplicate to minimize the test errors.

7.Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.

8.Avoid cross‐contamination by changing tips, using separate reservoirs for each reagent, avoid using the suction head without extensive wash.

9.Do not mix the reagents from different batches

10.Stop Solution should be added in the same order of the Substrate solution.

11.Chromogenic Substrate B is light‐sensitive, please avoid prolonged exposure to light.

12.The kit should be kept at 2 ‐ 8 °C  and cannot be used after expiration date. Standards to be used within 5 days may be stored at 2‐8℃ , otherwise Standards must be stored at ‐20 °C to avoid loss of bioactivity .

Result calculation

1. This standard curve is used to determine the amount of an unknown sample. Construct a standard curve by plotting the average O.D. (450 nm) for each standard on the vertical (Y) axis against the concentration on the horizontal (X) axis, and draw a best fit curve through the points on the graph.

2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the blank control before result interpretation. Construct the standard curve using graph paper or statistical software.

3. To determine the amount in each sample, first locate the O.D. value on the Y‐axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X‐axis and read the corresponding concentration.

4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.

5. Standard curve (reference in general, notforthis kitin particular):

 

Sensitivity and specificity

1. The sensitivity in this assay is 0.1ng/ml (Variable).

2. This assay has high sensitivity and excellent specificity for detection of analyte. No significant cross‐reactivity or interference between analyte and analogues was observed.

3.Storage:  2‐8 °C

4.Validity:  six months.

Direct ELISA

The direct detection method uses a labeled primary antibody that reacts directly with the antigen.Direct detection can be performed with antigen that is directly immobilized on the assay plate (Figure1) or by first attaching a capture antibody to the plate surface (Sandwich ELISA,Figure2).Atlast,the substrate will be added to complete colorimetric reaction by HRP‐conjugated‐antibody.

Figure 1

Figure 2

Competitive ELISAs

Competitive ELISA can be used to determine if a molecule is present in the sample or not,or to assess the concentration of antigen, hormone or small molecules including cAMP,cGMP,NO etc. First,the wells will be coated with secondary antibody. The primary antibody will be added to bind it.Then, molecules of interest (antigen) in sample will be added along with the HRP‐conjugated‐antigen.At last,the substrate will be added to initiate colorimetric reaction by HRP‐conjugated‐antigen(Figure3). The more intense the coloris,the less molecules of interest is present.This is because the free antigen molecule binds the primary antibody and competes away the HRP‐conjugated‐antigen. If there is not much of the molecule of interest,the HRP‐conjugated‐antigen will bind to the primary antibody,therefore, stay with the plate to enhance the color.

Figure 3

Your Analyte ELISA Kit

Intended use

The kit is used to detect the level of Your analyte in cell culture, serum blood plasma and other suitable sample solution.

Assay principle

The kit uses a double‐antibody sandwich enzyme‐linked immunosorbent assay (ELISA) to analyze the level of Your analyte in samples. First add sample to wells pre‐coated with one Your analyte antibody to capture availa ble analyte in solution, and perform incubating and washing procedures to remove unbo und substance. Then add second HRP‐conjugated Your analyte antibody to bind the captured analyte, followed by another round of incubation and washing procedures to remove unbound antibody‐ HRP. Finally, HRP substrates are added, incubated for detection, and a blue color is deve loped. Reaction is stopped and color turns to yellow when Stopping Solution (acidic ) is adde d. The yellow color intensity proportionally correlates to the concentration of the Your analyte in samples.

Manufactured by:

Novatein BiosciencesWest Cummings Park Woburn,MA 01801 USA

Telephone: (617) 238-1396 310

Materials supplied

1 Precoated Strip‐plate 12‐well X 8 strips 7 Substrate A 6 mL
2 Standard ( 40 ng/mL ) 0.6 mL 8 Substrate B 6 mL
3 20 X Wash Solution 20 mL 9 Stopping Solution 6 mL
4 Standard Diluent 6 mL 10 Instruction 1
5 Sample Diluent 6 mL 11 Plate Closure Membrane 1
6 HRP‐conjugated antibody 6 mL 12 Sealed Bag 1

Note: Suggested standard dilution to the following concentrations:Variables ng/ml.

Materials required but not supplied

1.37 °C incubator

2. Standard plate reader capable of measuring absorbance at 450 nm, with correction wavelength set between 540 nm and 570 nm.

3. Precision pipettes and disposable pipette tips

4. Distilled water

5. Disposable tubes or 96 ‐well plate for sample dilution

6. Multi‐channel pipettes, manifold dispenser or automated microplate washer.

7. Absorbent paper

Assay procedures

1.After taking out from 2‐8°C refrigerator, prewarm the kit 30 minutes at room temperature before using.

2. Make standard and sample dilutions in a clean 96 ‐ well plate. Dilute the 20 × Wash Solution to 1X Wash Solution.

3. Set standard wells, testing sample and blank wells on the assay plate/strip. Transfer diluted standard 50 μl to standard wells, diluted sample 50 μl to sample wells, sample diluent only to blank wells.

4. Incubate the plate for 30 minutes in a 37 °C incubator.

5. Decant as much liquid as possible, fill the wells with washing solution, oscillate the plate on a oscillating shaker if available for 1 mi n, decant the washing solution and remove residual liquid with absorbent paper. Repeat wash four times.

6. Add HRP ‐ conjugated antibody 50 μl to each well, except the blank well. Mix by shaking gently, and incubate for 30 minutes at 37 °C.

7. Wash the plate or strips as described in Step #5.

8. Add chromogenic Substrate A and B: Substrate A 50 μl and Substrate B 50 μl to each well. Mix gently, incubate for 15 min at 37 °C.

9. Add Stop Solution 50 μl immediately to each well to to p the reaction ( the blue color change to yellow ).

10. Measure the optical density (OD) at 450 nm within 15 min

11. Construct standard curve ( plotting the mean OD 450 for each standard on the Y ‐ axis against concentration on the X ‐ axis, draw a best ‐ fit log ‐ log curve through the points ) and calculate linear regression equation, then use sample OD values and regression equation to calculate the corresponding sample concen tration. It should be remembered that the sample has been diluted and its actual concentration should be justified by dilution factor(the measurem ent and calculation can be accomplished by software like SoftMax).

Specimen requirements

1. Can’t detect the samples containing NaN3 , since NaN3 inhibits HRP (horseradish peroxidase) activity.

2. If the specimen can not be tested immediately, it should be kept < ‐ 20 °C and repeated freeze /thaw should be avoided.

3. The samples should be cleared by extensive centrifugation to remove any particulates.

4. For serum samples, allow blood to clot for 2 hrs at room temperature before centrifuging For 20 minutes at 1000 X g. Remove serum for assay immediately or aliquot and store serum at < 20 °C.

5. For plasma, using EDTA or heparin as anti coagulant, spin for 20 minutes at 1000 X g within 30 minutes of collection. Assay immediately or aliquot and store serum at < 20 °C.

Important notes

1. The operation should be carried out in strict accordance with the provided instructions.

2. To preserve unused strip-wells, it sh ould be stored in the sealed bag.

3. Always avoid foaming when mixing or reconstituting protein solutions.

4. Pipette reagents and samples into the center of each well.

5. The samples should be transferred into the assay wells within 15 minutes of dilution.

6. We recommended that all standard, test samp les are tested in duplicate to minimize the test errors.

7. Please justify the results w ith dilution factor. Two dilution is recommended for each sample to get the best testing result.

8. If the blue color too shallo w after 15 minutes incubation with the substrates, it may be appropriate to extend the incubation time.

9. Avoid cross-contamination by changing tips, us ing separate reservoirs for each reagent, avoid using the suction head without extensive wash.

10. Do not mix the reagents from different batches

11. Stop Solution should be added in the sa me order of the Substrate solution.

12. Chromogenic Substrate B is light-sensitive, please avoid prolonged exposure to light. The kit should be kept at 2 – 8 °C and can not be used after expiration date. The Standard should be kept at < 20 °C after receiving.

Procedures in summary

Prewarm all reagents to roomtemperature before assay.

Prepare reagents, standard dilutions, sample dilutions in clean tubes or 96‐well plate.

Transfer standard and samples to assay plate/strip, incubate 30 minutes at 37 °C

Plate‐wash four times, add HRP‐conjugated antibody and incubate 30 minutes at 37 °C

Plate‐wash four times, add Substrate A and B, incubate 15 minutes at 37 °C

Add stop solution

Measure within 15 minutes

Calculation

Result calculation

Get the OD 450 mean value of the duplicate readings for each standard, control, and sample and subtract the average zero standard.

Create a standard curve using computer software capable of generating a log ‐log curve‐fit (Excel, for example). As an alternative, cons truct 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 gra ph. Obtain the linear regression equation, and calculate each sample concentration using this formula.

Typical standard curve

Standard Concentration ( pg /mL) Mean OD 450 Adjusted
0.00 0.042 none
31.20 0.073 0.03
62.50 0.109 0.06
125.00 0.173 0.13
250.00 0.313 0.271
500.00 0.581 0.539
1000.00 1.111 0.938
2000.00 2.148 2.106

Sample analyte concentration will be 10 x pg/mL, where X = (Y + 0.0041)/0.001, Y is the value of sample OD 450 .

Assay range:0.01–1000 ng/ml

Package size: 96 /T

Storage: 2‐8°C.

1. Analyte capture

Provided microplate is precoated with capture antibody(Ab). Any analyte ( red dots)in the standard or samples added to the well will be captured. Unbound molecules are washed away.

2. HRP-detection Ab binding to captured analyte

A second antibody conjugated with HRP (blue shield) is added and binds to the captured Analyte. Free detection antibody Is washed away.

3. HRP/substrate color display

HRP substrate TMB (Tetramethylbenzidine) is added to the wells. Blue color is developed proportionally to the amount of analyte in the sample. Blue color development is stopped and turns to yellow when the Stop Solution is added. OD450 is measured to make standard curve and calculate analyte concentration.

RECOMMENDED SAMPLE HANDLING AND PROCESSING

Blood, Plasma, and Serum

General blood sample processing requirements

Proper processing of the collected samples is critical. It is particularly important that time constraints are observed and that samples are not left at room temperature longer than necessary. Samples should be processed and frozen at ‐80 °C within 2 hours of collection.

Plasma

Centrifuge plasma tubes (Citrate, Heparin or EDTA tubes) at room temperature. If within tube manufacture’s specifications, spin at 2200 x g (not RPM) for 15 minutes (this speed has been chosen to attempt to remove all cellular contents and platelets from samples). Observe separation of blood cells and plasma, with plasma layer on top.  Draw off only the plasma layer. Take care not to disturb buffy coat when aliquoting by leaving some plasma behind and avoiding the cell layer. Aliquot into appropriately labeled tubes. Aliquot samples immediately and then place aliquoted samples in a ‐80 °C freezer.

Note: Plasma samples do not need to clot, and should be centrifuged immediately after collection.

Serum

Allow serum to clot for 60 minutes at room temperature prior to centrifugation. Centrifuge serum tubes. If within tube manufacture’s specifications, spin at 2200 x g (not RPM) for 15 minutes (this speed has been chosen to attempt to remove all cellular contents and platelets from samples). Observe separation of blood cells and serum, with serum layer on top. Draw off only the serum and aliquot into appropriately labeled tubes. Aliquot samples immediately and then place aliquoted samples in a ‐80 °C freezer.

Urine

Collect neat urine sample. Clarify the urine by centrifugation at 14,000 x g for 5 minutes, prior to freezing, and collect the clarified supernatant. Store aliquots at ‐80 °C.

Cell culture lysate

Collect samples using Mammalian Protein Extraction Reagent (Thermo Scientific) following manufacturer instructions. Sufficient material can usually be obtained from a cell monolayer, 80-100% confluent, in a single well of a six‐well plate, harvested with 300 μL lysis buffer.

To harvest cell lysate: -Wash cells three (3) times with Dulbecco’s Phosphate Buffered Saline (DPBS) prior to lysing. Add protease inhibitor cocktail to the lysis buffer to inhibit protease activity. Add lysis buffer to the cells followed by appropriate lysis procedure. Centrifuge lysed cells at 14,000 x g for 5 minutes, and collect the supernatant (clarified lysate). Quantify total protein amount using BCA Protein Assay Kit or similar protein quantification method.

Cell conditioned media (Cell culture supernatants)

For studies with cells cultured in 1‐10% fetal bovine serum, consider including proper control samples (media controls, untreated cells and/or vehicle‐treated cells) depending on the scientific question to be addressed. Keep the media volume to a minimum in order to increase protein concentration, and strive to have cell density at 75% surface area or greater. Sufficient material can usually be obtained from 1 mL media removed from 80‐100% confluent cell monolayer from a single well of a six‐well plate. Time points of less than 24 hours may be too early to show a differential signal, consider reducing the volume of media used for these types of experiments. Clarify cell supernatant by centrifugation at 14,000 x g for 5 minutes, prior to freezing, and collect the clarified supernatant. The minimum volume required of clarified supernatant is 100 μL. Store samples in a ‐80 °C freezer.

Tissue or xenograft tumor homogenates

Snap freeze (at least 5 mg) excised tissue in liquid nitrogen within 5‐10 minutes of excision. Pulverize frozen tissue (using a freezer mill or similar) while maintaining low temperature using liquid nitrogen or dry ice. Use T-Per tissue protein extraction agent (Thermo Scientific) per manufacturer’s recommendation. Add 200 uL extraction buffer plus protease inhibitor cocktail per 10 mg of tissue. Homogenize in tube on ice with rotary pestle for 30 seconds, until no tissue fragments are visible.  Centrifuge while cold at 14,000 x g for 10 minutes. • Collect supernatant (keep on ice). • Filter through a 0.2 micron filter into a sterile tube or plate. • Quantify total protein amount using BCA Protein Assay Kit or other similar protein quantification method.

Sample Size Requirements

Depends on the analyte to be assayed. Please contact technical@stratech.co.uk

ELISA Troubleshooting

Probable Cause:Solution/ Action
High incubation temperature:Incubate at room temperature (25 oC) throughout the procedure
Insufficient washing of the plate:Fill the wells with wash buffer and aspirate completely for the next wash
Increase the number of washes
Add soak time (20-30 seconds) in between the washes
Use automated plate washer, if available and check that all the channels are operating properly
Concentrated streptavidin-HRPStreptavidin-HRP was not diluted properly
Dilute the streptavidin-HRP as mentioned in the manual
Light exposure during substrate incubationThe TMB substrate is light sensitive and turns to blue color in the presence of light. The incubation must be carried out in dark.
Stop solution not addedColor will continue to develop if stop solution is not added
Diluents came with the kit were not usedStandards/ sample, detection antibody and streptavidin-HRP must be diluted in the respective buffers came with the kit. Do not use buffers from other kits
Contaminated solutionsPrepare fresh working solutions
Probable Cause:Solution/ Action
Improper standard reconstitution:Spin the vial briefly before opening
Reconstitute the standard as mentioned in the manual. After reconstitution, leave it atleast for 10 minutes at room temperature
Do not store and reuse diluted standards
Curve fitting problem:Log transform the values on both axes
Use 4-PL/ 5-PL curve fitting programs
Incubation temperature/ timeUse the recommended standard incubation conditions
Poor dilutionsPipetting error. Check pipetting technique and calculations.
Use calibrated pipettes.
Probable Cause:Solution/ Action
Omission of reagent(s):Read the manual entirely. Check that all the reagents are added in the correct order as stated in the manual
Incorrect detection antibody was used:Use the detection antibody came with the kit
Chromogen solutions were mixed improperlyUse the recommended procedure to prepare the TMB substrate
HRP inhibitor in sample/ buffersCheck that the samples/ buffers do not have sodium azide as it will inhibit peroxidase reaction.
Vigorous washingIf the washing is done manually, pipette the wash buffer gently.
Dried wellsDo not allow the wells to dry out during the assay. Seal with the supplied adhesive cover during incubations
Improper plate reader settingsCheck the wavelength and read the plate again
Probable Cause:Solution/ Action
Insufficient washing of the plateFill the wells with wash buffer and aspirate completely for the next wash
Increase number of washes
Add soak time (20-30 seconds) in between the washes
Use automated plate washer, is available and check that all the channels are functioning properly
Poor dilutionsPipetting error. Check pipetting technique and calculations.
Use calibrated pipettes.
Improper mixing of samples/ buffersMix the samples well before pipetting
Thoroughly mix the working solutions of detection antibody/ streptavidin-HRP
Contamination from other wellsDo not reuse the adhesive covers from previous assay setups
Change pipette tips during reagent addition. If same pipette tip is being used to dispense reagents, care should be taken, not to touch the solution in the well
Precipitates in the samples/ bufferIf precipitates are visible in wash buffer concentrate, keep it at 37 oC for 10-15 minutes until no precipitates are visible
Centrifuge the samples to remove particulate matter
Dried wellsDo not allow the wells to dry out during the assay. Seal with the supplied adhesive cover during incubations

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