Our cell separation columns are designed as a rapid, one step tool to enrich for specific cell populations. We have attempted to make the columns simple and user friendly. To ensure that all users get maximal efficiency from these columns, we have summarized some common problems below. We attempt to identify the likely sources of these problems and possible courses of action to be taken to avoid them. We urge users to review these tips and if additional advice is required to contact our technical service department.

Additional Information:

• Whole Blood Lyse Kit
  • Erythrocyte Lysing Kit
• Flow Cytometry Kits

Problem: Column buffer does not drip after removal of bottom cap

Possible Source	Test or Action
Air lock in column tip	Tap side of column to dislodge air bubble

Problem: Very slow flow rate following cell loading

Possible Source	Test or Action
Cell clumps and debris have accumulated on the top white filter	Remove cell debris that has accumulated on the white filter with a sterile pipette
	Reduce the amount of time single cells incubate in a small volume as it may reduce the tendency of cells to form clumps

Problem: Cell recovery low

Possible Source	Test or Action
Poor specimen preparation	Ensure that suspension contains single cells with no clumps and minimal RBC
Column was either overloaded or underloaded	Load the optimal number of total cells indicated in the product insert
Improper calculation	Percent recovery should be calculated by dividing the number of target cells recovered by the number of target cells loaded

Problem: Cell purity is low

Possible Source	Test or Action
Non-optimal number of total cells loaded	Reduce the total number of cells loaded and determine by immunophenotype if the frequency of the contaminating population is reduced
Poor specimen preparation or tissue source	High frequency of dead cells in recovered sample. Determine cell viability and perform immunophenotype analysis gating only on live cells

ELISA Development

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• High background
• No signal
• Too much signal - whole plate turned uniformly blue
• Standard curve achieved but poor discrimination between points
• Poor duplicates
• Poor assay to assay reproducibility
• No signal when a signal is expected, but standard curve looks fine
• Samples are reading too high, but standard curve looks fine
• Very Low Readings Across the Plate
• Green color develops upon addition of stop solution when using streptavidin-HRP
• Edge Effects
• Drift

Problem: High Background

Possible Source	Test or Action
Insufficient washing	See washing procedure on page 4 of theELISA Development Guide
	Increase number of washes Add a 30 second soak step inbetween washes

Problem: No signal when a signal is expected

Possible Source	Test or Action
Reagents added in incorrect order, or incorrectly prepared	Repeat assay Check calculations and make new buffers, standards, etc. Review protocol
Contamination of HRP with	Use fresh reagents
Not enough antibody used	Increase concentration
Standard has gone bad (if there is a signal in the sample wells)	Check that standard was handled according to directions. Use new vial.
Buffer containing FCS used to reconstitute antibodies	Requalify your reagents of choice
Capture antibody did not bind to plate	Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein
Buffers contaminated	Make fresh buffers

Problem: Too much signal - whole plate turned uniformly blue

Possible Source	Test or Action
Insufficient washing/washing step skipped - unbound peroxidase remaining	See washing procedure on page 4 of theELISA Development Guide
Substrate Solution mixed too early and turned blue	Substrate Solution should be mixed and used immediately
Too much streptavidin-HRP	Check dilution, titrate if necessary
Plate sealers or reagent reservoirs reused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically	Use fresh plate sealer and reagent reservoir for each step
Buffers contaminated with metals or HRP	Make fresh buffers

Problem: Standard curve achieved but poor discrimination between points (low or flat curve)

Possible Source	Test or Action
Not enough streptavidin-HRP	Check dilution, titrate if necessary
Capture antibody did not bind well to plate	Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein
Not enough detection antibody	Check dilution, titrate if necessary
Plate not developed long enough	Increase Substrate Solution incubation time Use recommended brand of Substrate Solution
Incorrect procedure	Go back to General ELISA Protocol; eliminate modifications, if any
Improper calculation of standard curve dilutions	Check calculations, make new standard curve

Problem: Poor Duplicates

Possible Source	Test or Action
Insufficient washing	See washing procedures on page 4 of theELISA Development Guide If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash
Uneven plate coating due to procedural error or poor plate quality (can bind unevenly)	Dilute in PBS without additional protein  Check coating and blocking volumes, times and method of reagent addition. Check plate used  Use an ELISA plate (not a tissue culture plate)
Plate sealer reused	Use a fresh plate sealer for each step
No plate sealers used	Use plate sealers
Buffers contaminated	Make fresh buffers

Problem: Poor assay to assay reproducibility

Possible Source	Test or Action
Insufficient washing	See washing procedure on page 4 of theELISA Development Guide  If using an automatic plate washer, check that all ports are clean and free of obstructions
Variations in incubation temperature	Adhere to recommended incubation temperature  Avoid incubating plates in areas where enviromental conditions vary
Variations in protocol	Adhere to the same protocol from run to run
Plate sealer reused, resulting in presence of residual HRP which will turn the TMB blue	Use fresh plate sealer for each step
Improper calculation of standard curve dilutions	Check calculations, make new standard curve  Use internal controls
Buffers contaminated	Make fresh buffers

Problem: No signal when a signal is expected, but standard curve looks fine

Possible Source	Test or Action
No cytokine in sample	Use internal controls  Repeat experiment, reconsider experimental parameters
Sample matrix is masking detection	Dilute samples at least 1:2 in appropriate diluent, or preferably, do a series of dilutions to look at recovery

Problem: Samples are reading too high, but standard curve looks fine

Possible Source	Test or Action
Samples contain cytokine levels above assay range	Dilute samples and run again

Problem: Very low readings across the plate

Possible Source	Test or Action
Incorrect wavelengths	Check filters/reader
Insufficient development time	Increase development time
Coated plates are old and have gone bad	Coat new plates
Capture antibody did not bind to the plate	Use an ELISA plate (not a tissue culture plate)  Dilute in PBS without additional protein
Buffer containing FCS used to reconstitute antibodies	Requalify your reagents of choice

Problem: Green color develops upon addition of stop solution when using streptavidin-HRP

Possible Source	Test or Action
Reagents not mixed well enough in wells	Tap plate

Problem: Edge Effects

Possible Source	Test or Action
Uneven temperatures around work surface	Avoid incubating plates in areas where environmental conditions vary
	Use plate sealers

 

Possible Source	Test or Action
Interrupted assay set-up	Assay set-up should be continuous - have all standards and samples prepared appropriately before commencement of the assay
Reagents not at room temperature	Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts

Problem: Drift

Possible Source	Test or Action
Interrupted assay set-up	Assay set-up should be continuous - have all standards and samples prepared appropriately before commencement of the assay
Reagents not at room temperature	Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts

ELISA failure can be attributed to many factors. Many errors can be avoided if the protocol is read and fully understood before starting the assay. On identifying assay failure, check the expiration dates of the individual reagents and ensure that all the reagents have been stored as indicated on the product label. Once this has been established, check for signs of instability or deterioration in reagent solutions, (e.g., precipitation or discoloration). All substrate solutions should be colorless. Whenever possible, use clean plastic disposable pipettes, tips, and containers for reagent preparation and storage. Avoid cross-contamination of kit reagents by changing pipette tips between addition of each standard, sample and reagent. Finally, ensure that specified incubation times and temperatures have been adhered to and that no substitution of kit reagents has occurred. To improve accuracy, it is recommended that samples and standards be run in duplicate. For further assistance, please contact our technical service department.

Problem: Poor Precision

Possible Source	Test or Action
Incomplete washing of wells	Ensure that wash apparatus is working correctly
Inadequate aspiration of wells	Wells should appear dry after aspiration
Unequal mixing of reagents	Ensure adequate mixing
Pipetting error	Repeat assay

Problem: Poor Standard Curve

Possible Source	Test or Action
Improper dilution of highest standard, standard curve, or blank values*	Ensure use of appropriate diluent as blank value. Ensure accurate completion of 2-fold dilution series* **
Incomplete washing of wells	Ensure that wash apparatus is working correctly
Inadequate aspiration of wells	Wells should appear dry after aspiration
Unequal volumes added to wells	Check pipette function; recalibrate if necessary
Color Reagents A and B mixed too early*	Substrate Solution must be used within 15 minutes of mixing*
Pipetting error	Repeat assay

Problem: Inadequate Color Development

Possible Source	Test or Action
Inadequate volume of substrate added to wells	Check pipette function
Incomplete mixing of Color Reagents A and B*	Ensure that equal volumes of Color Reagents A and B are properly mixed*
Incorrect incubation times or temperature	Adhere to recommended incubation times and temperatures
Conjugate or color reagent failure	Check by mixing equal volumes of Color Reagents A and B and conjugate. Color should develop immediately*

Problem: Edge Effects

Possible Source	Test or Action
Uneven temperatures around work surface	Adhere to recommended incubation periods and temperatures
Inadequate fixing of plate cover, leading to evaporation	Ensure correct use of plate cover

Problem: Drift

Possible Source	Test or Action
Interrupted assay set-up or reagents not at room temperature	Assay set-up should be continuous. Have all standards/samples prepared appropriately before commencement of the assay

Problem: Unexpected Result

Possible Source	Test or Action
We as manufacturers take great care to ensure that our products are suitable for use with all valid samples, as designated in the product inserts. However, it is possible that in some cases high levels of interfering factors may cause unusual results.

ELISpot

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Observation	Problem	Corrective Action
Following the incubation with BCIP/NBT chromogen and rinsing the microplate with deionized water, the dark-blue background color of filter membrane attenuates visualization and quantitation of spots.	Wet membrane	Microplates cannot be analyzed accurately until PVDF filter membranes are completely dry. Wait until membrane becomes dry, usually 15 - 30 minutes at 37° C or 60 - 90 minutes at room temperature
The number of spots in the wells that contained the cells is high but their contrast as well as intensity of staining in the Positive Control wells is low.	Underdevelopment - may be a result of using Streptavidin-AP and/or BCIP/NBT solutions that have not been brought to room temperature	Bring reagents to room temperature before adding to the wells.
The number of spots in the wells that contained cells is lower than expected whereas Positive Control wells turned black-blue.	Cell stimulation problem	Ensure that reagents used to stimulate the cytokine release from the cells retained their biological activity. One way to check is to perform immunocytochemistry on fixed cells after stimulation.
	Too few cells added to the wells	Increase the number of cells added per well.
Following incubation with BCIP/NBT and drying the microplate, the density of the spots makes it difficult to quantify them.	Too many cells were added to the wells	Make dilutions of cells (i.e., 1 x 106, 5 x 105, 1 x 105, 5 x 104, 1 x 104 cells per well) to determine the optimal number of cells that will result in formation of distinct spots.

Fluorokine® Receptor Detection Kits are designed as alternative reagents for the detection of cell surface cytokine receptors by flow cytometry. Although the staining procedure of cells with this line of reagents is straight forward, some situations can present difficulties in data interpretation. In the table below, we attempt to point out some common problems that users of Fluorokine Receptor Detection Kits may encounter. The variable nature of cytometry instrumentation and instrument set-up can dramatically influence the quality of data generated with these reagents.

Cell surface receptor analysis involves the monitoring of protein structures that are in a dynamic equilibrium with their environment so changes in the medium such as pH, temperature, ionic strength, or presence of other proteins and/or cytokines will ultimately influence the binding properties of each labeled ligand. Lastly, the biological activity of labeled ligands can modulate a cell’s receptor density. We hope the tips below will help investigators overcome simple obstacles, but if additional help is required, we encourage you to contact our technical service department.

Problem: Cells do not stain with Fluorokine

Possible Source	Test or Action
Cell processing error	Check all steps of the staining protocol to ensure proper cell processing
Cells were left on ice too long and receptors may have been downregulated	Stain fresh cells
Cells may have lost or decreased receptor numbers during culture	Use another receptor positive cell line to test reagent or retest same cells at different cell cycle stages

Problem: Fluorokine stains known receptor negative cells

Possible Source	Test or Action
Voltage being applied to photomultiplier tubes on cytometer are set too high	Reduce PMT voltage until >95% of events fall in the first decade of the log scale
Concentration of Fluorokine in the reaction is too high	Dilute the Fluorokine and compare signal intensity against cells that do express receptors
Transfected cells have upregulated other cell surface structures	Test Fluorokine on non-transfected cells or on other transfectants using a different parent line

Problem: Blocking antibody does not give 100% inhibition of Fluorokine signal

Possible Source	Test or Action
Cells were not pre-treated with Ig to block Fc receptors	Repeat the test by first treating cells with mouse or human Ig to block Fc receptors

Problem: Inhibition of Fluorokine staining with unconjugated cytokine is not 100%

Possible Source	Test or Action
Fluorokine present at too high concentration	Repeat reaction using less Fluorokine
Addition of unconjugated cytokine upregulated receptors	Perform all reactions at 2 - 8° C and in the presence of metabolic blockers, e.g.: azide, cytochalasin B, etc.
Numerically the inhibition is not 100%	Comparison of mean fluorescence intensity between the two conditions may reveal substantial inhibition

Immunohistochemistry

Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also require troubleshooting a variety of factors. The tables below highlight common IHC/ICC issues and provide appropriate experimental actions. For further assistance, please contact our technical service department.

Problem: Lack of Staining

Possible Source	Test or Action
Lack of antigen.	Check protein expression by in situhybridization (in some rare cases translation may be blocked even though mRNA is detected).
Antibodies do not work due to improper storage.	Follow storage instructions on the datasheet. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) and avoid repeated freeze-thaw cycles.
Inactive primary or secondary antibodies.	Test reporter system independently to assess reagent viability.
Inadequate tissue fixation.	Try increasing the fixation time or try a different fixative.
Tissue overfixation.	Reduce the duration of the immersion or post-fixation steps. If immersion fixation cannot be avoided (for example, collection ofpostmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents.
Incompatible secondary and primary antibodies.	Use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody.
Antigen was destroyed before incubation with the primary antibody.	If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody.
Epitope altered during fixation or embedding procedure.	Try restoring immunoreactivity through various antigen retrieval techniques.  Embed tissue at 58 °C or below.
Antigen retrieval was ineffective.	Increase the time of treatment or change the treatment solution.
Reagents omitted or used in wrong order.	Repeat staining and confirm that correct reagents are used and are added in the correct order.

Problem: High Background

Possible Source	Test or Action
High concentration of primary and or secondary antibodies.	Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.
Hydrophobic interactions of the antibody and proteins in the tissue.	Lower the ionic strength of the antibody diluent (particularly monoclonal antibodies respond well to reducing the salt concentration).
Non-speci?c binding of primary and/or secondary reagents to tissues.	Use blocking step just prior to primary antibody incubation (we use 1% bovine serum albumin with 10% normal donkey serum). Non-fat dry milk is another option.
Non-specific binding of secondary antibody.	Use an antibody that has cross-reactive IgG species removed (absorbed against sample species).
Tissue dried out.	Avoid letting the tissue dry during the staining procedure.
Reagents sticking to old or poorly prepared slides.	Start over with freshly prepared or purchased slides.
Background due to ionic interactions.	Increase the ionic strength of the diluent buffer.

Problem: Cell/Tissue Morphology is Destroyed

Possible Source	Test or Action
Antigen retrieval methods are too harsh.	Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen.
Tissue sections falling off slide. (more common with frozen sections)	Increase fixation time. Empirically determine an additional or alternative fixative.  Use freshly prepared, adequately charged slides.
Tissue section appears torn or folded. Air bubbles under section.	Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results.
Poor resolution of tissue morphology	Cut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections.
Underfixation has physically damaged the tissue or cells during the staining process	Increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio.  Cut smaller pieces of tissue for more thorough immersion fixation.
Autolysis of tissue leading to staining of necrotic debris.	Increase the fixation time, ratio. Consider using cross-linking fixative.

Problem: Staining is Inappropriate

Possible Source	Test or Action
Fixation method is inappropriate for the antigen.	Try a different fixative or increase the fixation time.
Antigen retrieval may be inappropriate for this antigen or tissue.	Try different antigen retrieval conditions.
Electrostatic charge of the antigen has been altered.	Try adjusting the pH or cation concentration of the antibody diluent.
Delay in fixation caused diffusion of the antigen.	Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative.

Our MagCellect Cell Isolation kits are designed to rapidly isolate specific cell populations. We have attempted to make the kits complete and easy to use. To ensure that all users get optimal performance with these products we have summarized some common problems below. We attempt to identify the likely sources of these problems and possible courses of action to be taken to avoid them. We urge users to review these tips and if additional advice is required to contact our technical service department.

Problem: Low cell recovery

Possible Source	Test or Action
Excessive or nonspecific binding of antibodies and/or ferrofluids to the desired cells	Follow the incubation times indicated and/or carry out reactions at the temperature indicated in the product insert
Non optimal number of cells used	Use the recommended ratio of cells and antibody mixture

Problem: Low/poor cell purity

Possible Source	Test or Action
Insufficient antibody used	Use the recommended ratio of cells and antibody mixture
Non-optimal amounts of ferrofluid used	Use the recommended ratio of cells and ferrofluids
Carryover of magnetically tagged cells in a negative selection procedure	Harvest un-tagged cells carefully without touching the tube wall where magnetically tagged cells are located
Magnetic cell separation reaction had not gone to completion	Perform the magnetic separation with the recommended magnet and for the recommended time

MycoProbe® Mycoplasma Detection Kit

For further assistance, please contact our technical service department.

Observation: High background level

Problem	Corrective Action
Insufficient washing	Wash per protocol being sure to remove all Wash Buffer from wells before addition of next component
Contamination with alkaline phosphatase	Keep work area clean and free of alkaline phosphatase

Observation: Poor precision

Problem	Corrective Action
Plate not washed before use	Wash per protocol
RNase contamination	Use RNase-free technique
Pipetting error	Use a new pipet tip for each pipetting step and use proper technique

Observation: No signal for positive control

Problem	Corrective Action
Component or step omitted	Read protocol thoroughly before repeating the assay
RNase contamination	Use RNase-free technique

Proteome Profiler Array Kits

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Observation	Problem	Corrective Action
No signals on positive control spots	Concentration of detection antibody and/or SA-HRP too low	Use concentration/dilution specified
	Chemiluminescent reagents failed	Repeat with fresh chemiluminescent reagents according to manufacturers protocol
	Inadequate exposure time to film	Increase exposure time
No or low signals on target spots	Sample dilution too high	Use more sample
	Analyte abundance in sample is low	Use more sample
		Use different cell line
		Verify that conditions used to stimulate cells were optimal
	Sample deteriorated during preparation	Supplement buffers with protease and phosphatase inhibitors where indicated
	Sample deteriorated during storage	All samples should be stored at ≤ 70 °C.
		Avoid freeze-thaw cycles
	Inadequate exposure time to film	Increase exposure time
Signals on negative control spots	Concentration of detection antibody and/or SA-HRP too high	Use concentration/dilution specified
	Sample concentration too high	Use less sample
Uneven or high background on blank areas of array	Insufficient washing	Perform number of washes with volume indicated as specified in the product insert protocol
		Perform washes in large container and not the 4-Well Multi-dish
	Concentration of detection antibody and/or SA-HRP too high	Use concentration/dilution specified
	Array was allowed to dry out partially during the procedure	Always keep arrays submerged. Minimize time the array is exposed to air.
	Creased or scratched arrays	Use flat-tipped forceps to handle arrays on array number.
Too many spots have signals	Insufficient washing	Perform washes as stated in product insert. Perform washes in a large container, not in the 4-Well Multi-dish.
	Concentration of detection antibody and/or SA-HRP too high	Use concentration/dilution specified
	Sample concentration too high	Use less sample

Proteome Purify™ Immunodepletion Kits

For further assistance, please contact our technical service department.

Problem: Incomplete or no flow through spin filter

Cause	Solution
Cap on too tight	Loosen or remove spin filter cap.
Air lock	Tap side of spin filter to dislodge air bubble
Centrifuge parameters need to be adjusted	2 minutes at 1000-2000 x g usually sufficient.

Western Blot

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• Observation: No Bands Observed
• Observation: Faint Bands (Weak Signal)
• Observation: Extra Bands
• Observation: High Background
• Observation: Diffuse Bands
• Observation: White Bands (ECL method)
• Observation: Patchy uneven spots all over the blot

Observation: No Bands Observed

Possible Source	Suggestion
Insufficient antibody	Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).
	Antibody may have lost activity. Perform a Dot Blot.
Insufficient protein	Increase the amount of total protein loaded on gel.
	Confirm the presence of protein by another method.
	Use a positive control (recombinant protein, cell line or treat cells to express analyte of interest).
	Perform a Dot Blot.
Poor transfer	Wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer.
	Ensure that there is good contact between PVDF membrane and gel.
Incomplete transfer	Optimize transfer time. High MW protein may require more time for transfer.
	To ensure transfer is complete, stain the membrane with Ponceau S, Amido Black or India Ink.
	Use prestained MW marker (R&D Systems Catalog # MW002 or equivalent).
Over transfer	Reduce voltage or time of transfer for low molecular weight proteins (< 10 kDa).
Isoelectric point is >9	Use alternative buffer system with higher pH such as CAPS (pH 10.5).
Incorrect secondary antibody used	Confirm host species and Ig type of primary antibody.
Old antibody	If antibody is expired or past manufacturer warranty, purchase fresh antibody.
Incorrect storage of antibodies	Follow manufacturer's recommended storage and avoid freeze/thaw cycles.
Sodium Azide contamination	Make sure buffers do not contain Sodium Azide as this can quench HRP signal.
Insufficient incubation time with primary antibody	Extend incubation time to overnight at 4°C.

Observation: Faint Bands (Weak Signal)

Possible Source	Suggestion
Low protein-antibody binding	Reduce the number of washes to minimum.
	Reduce NaCl concentration in Blotting Buffer used for wash steps (recommended range 0.15M - 0.5M).
	Reduce NaCl concentration in Antibody Solution (recommended range 0.15M - 0.5M).
Insufficient antibody	Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).
Insufficient protein	Increase the amount of total protein loaded on gel.
Inactive conjugate	Mix enzyme and substrate in a tube. If color does not develop or, it is weak. Make fresh or purchase new reagents. Switch to ECL.
Weak/Old ECL	Purchase new ECL reagents.
Non-fat dry milk may mask some antigen	Decrease milk percentage in Block and Antibody Solutions or substitute with 3% BSA.

Observation: Extra Bands

Possible Source	Suggestion
Non-specific binding of primary antibody	Reduce primary antibody concentration. (SeeFigure 1)
	Reduce the amount of total protein loaded on gel.
	Use monospecific or antigen affinity purified antibodies (such as R&D Systems "MAB" or "AF" designated antibodies).
Non-specific binding of secondary antibody	Run a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.
	Use monospecific or antigen affinity purified antibodies (such as R&D Systems "BAF" or "HAF" designated secondary antibodies).
Non-specific binding of primary or secondary antibodies	Add 0.1 - 0.5% Tween® 20 to primary or secondary Antibody Solution.
	Use 2% non-fat dry milk in Blotting Buffer as a starting point to dilute primary and secondary antibodies. Adjust antibody concentration up or down as needed. (See Figure 2a & 2b)
	Increase number of washes.
	Increase NaCl concentration in Blotting Buffer used for antibody dilution and wash steps (recommended range 0.15M - 0.5M). (SeeFigure 2c)
	Increase Tween® 20 concentration in Blotting Buffer used for wash steps (0.1%-0.5%).
Aggregation of analyte	Increase amount of DTT to ensure complete reducing of disulfide bonds (20 -100mM DTT). Heat in boiling water bath 5-10 minutes before loading onto gel.
	Perform a brief centrifugation.
Degradation of Analyte	Minimize freeze/thaw cycles of sample.
	Add protease inhibitors to sample before storage.
	Make fresh samples.
Contamination of reagents	Check buffers for particulate or bacterial contamination. Make fresh reagents.

Observation: High Background

Possible Source	Suggestion
Non-specific binding of primary antibody	Use monospecific or antigen affinity-purified antibodies (such as R&D Systems "MAB" or "AF" designated antibodies).
	Block in 5% milk. Adjust the milk (2-5%) or NaCl (0.15-0.5M) concentrations of primary Antibody Solution. (See Figure 3).
	Decrease antibody concentration.
Non-specific binding of secondary antibody	Run a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.
Insufficient blocking	Start with 5% dry milk with 0.1%- 0.5% Tween 20, 0.15 -0.5M NaCl in 25mM Tris (pH 7.4). Incubation time may be extended. Adjust milk concentration up or down as needed.
	Overnight blocking at 4°C may decrease blocking efficiency since detergents might not be effective at lower temperatures.
Non-fat dry milk may contain target antigen	Substitute with 3% BSA.
Non-fat dry milk contains endogenous biotin and is incompatible with avidin/streptavidin	Substitute with 3% BSA.
Some IgY antibodies may recognize milk protein	Substitute with 3% BSA.
Insufficient wash	Increase number of washes.
	Increase Tween 20 concentration in Wash Buffer (0.1%-0.5%).
Non-Specific Binding of Primary Antibody	Increase NaCl concentration in primary Antibody Solution and Blotting Buffer used for dilution of primary antibody and wash steps (recommended range 0.15M - 0.5M). (SeeFigure 4).
Film overexposed	Reduce exposure time.
	If target signal is too strong wait 5-10 minutes and re-expose to film.

Observation: Diffuse Bands

Possible Source	Suggestion
Excessive protein on gel	Reduce amount of protein loaded

Observation: White Bands (ECL method)

Possible Source	Suggestion
Excessive signal generated	Reduce antibody or protein concentration. Excessive antibody or protein can cause extremely high levels of localized signal (usually at a single band). This results in rapid, complete consumption of substrate at this point. Since there is no light production after the completion of this reaction, white bands are the result when exposed to film.

Observation: Patchy uneven spots all over the blot

Possible Source	Suggestion
Contamination of reagents	Check buffers for particulate or bacterial contaminate. Make fresh reagents.
Not enough solution during incubation or washing	Make sure membrane is fully immersed during washes and antibody incubations.
Air bubble trapped in membrane	Gently remove any air bubbles. Especially during transfer.
Uneven agitation during incubations	Ensure uniform agitation by placing on a rocker/shaker.
Contaminated equipment	Make sure that the electrophoresis unit is properly washed. Protein or pieces of gel remaining on the unit may stick to the membrane. Wash membrane thoroughly.
HRP aggregation	Filter conjugate to remove HRP aggregates.
Long exposure	Reduce exposure time.

Western Blot Figures

Figure 1 – Effect of Varying Primary Antibody Concentration:

Western blot optimization using R&D Systems rabbit anti-human/mouse/rat RSK1 affinity purified polyclonal antibody (Catalog # AF992). Human HeLa cells were solubilized in SDS gel buffer, and lysate from 1 x 105 cells per lane was resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Bolded concentrations indicate the reduction of the primary antibody concentration. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl. Blocked in 5% milk, 0.2 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.

 

Figure 2 – Effect of Varying Primary Antibody Concentration, % Milk in Antibody Solution, and NaCl Concentration in Blotting Buffer:

Western blot optimization using R&D Systems rabbit anti-human PTP1B affinity-purified polyclonal antibody (Catalog # AF1366). Human HeLa (Lane 1), MCF-7 (Lane 2), and TF-1 (Lane 3) cells were solubilized in SDS gel sample buffer, and lysates from 1 x 105 cells per lane were resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

	Figure 2a – Effect of Varying Primary Antibody Concentration in Antibody Solution with 2% Milk: Bolded concentrations indicate the dilution of the primary antibody concentration. Blocked in 5% milk,1.0 microgram/mLprimary antibody in 2% milk and 0.15 M NaCl. Blocked in 5% milk,0.3 microgram/mLprimary antibody in 2% milk and 0.15 M NaCl. Blocked in 5% milk,0.1 microgram/mLprimary antibody in 2% milk and 0.15 M NaCl.
	Figure 2b – Effect of Varying Primary Antibody Concentration in Antibody Solution with 5% Milk: In addition to the dilution of the primary antibody concentration (bolded), the milk concentration in the Antibody Solution was also increased. Blocked in 5% milk,1.0 microgram/mLprimary antibody in 5% milk and 0.15 M NaCl. Blocked in 5% milk,0.3 microgram/mLprimary antibody in 5% milk and 0.15 M NaCl. Blocked in 5% milk,0.1 microgram/mLprimary antibody in 5% milk and 0.15 M NaCl.
	Figure 2c – Effect of Varying NaCl Concentration in Blotting Buffer: Bolded concentrations indicate the increased NaCl concentrations in the Blotting Buffer. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 MNaCl. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.5 MNaCl.

 

 

Figure 3 – Effect of Varying Milk and NaCl Concentrations:

Western blot optimization using R&D Systems rabbit anti-human PTP1B affinity-purified polyclonal antibody (Catalog # AF1366). Human HeLa (Lane 1), MCF-7 (Lane 2), and TF-1 (Lane 3) cells were solubilized in SDS gel sample buffer, and lysates from 1 x 105 cells per lane were resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Bolded concentrations indicate adjustments in the milk and NaCl concentrations. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 2% milk and 0.15 M NaCl. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.5 M NaCl.

 

Figure 4 – Effect of Varying NaCl Concentration in Blotting Buffer:

Western blot optimization using R&D Systems rabbit anti-human/mouse/rat RSK1 affinity purified polyclonal antibody (Catalog # AF992). Human HeLa cells were solubilized in SDS gel buffer, and lysate from 1 x 105 cells per lane was resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Bolded concentrations indicate an increase in the NaCl concentration in the primary Antibody Solution. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.5 M NaCl.