Frequently Asked Antibody Troubleshooting Questions
Reconstitute in an equivalent volume to the weight you purchased, so that the final concentration is 1 mg/ml. For example, if you purchased 100 µg of antibody, resuspend in 100 µl of pure distilled water. Dissolve the entire contents in water by mixing and lightly centrifuging at room temperature. It is possible to dissolve the contents in larger volume (200-500 µl) but the subsequent dilution may have to be adjusted. Insoluble lipoproteins may aggregate on storage. These do not interfere with the solubility of the antibody, spin down and use the clear supernatant.
Antibodies diluted as directed are very stable. Months and probably years. One study has sown extended shelf life for commercial reagents, beyond the manufacturer's expiration date. For antibodies used very diluted against low level antigens (<2K molecules/cell) is probably safer make fresh solutions monthly.
Each freezing cycle causes aggregation of the immunoglobulin and loss of titer. Avoid freezing aliquots that you expect to use out in the next couple of years, unless they are small aliquots which you will use completely within 1 or 2 experiments. You may freeze reference reagents in small aliquots for reference or reagents to be stored without preservative.
Incubation for too short will not produce adequate signal. Incubation for too long can result in negative (unspecific) staining. Since the final result of your technique can only be determined at the end of your technique when no corrections can be made, the dilution and time of incubation for each antibody should be determined individually. In general, antibodies with known high affinity should be used at high dilution and overnight incubations. Antibodies with various affinities (polyclonal) must be experimented with to determine optimum time and dilution.
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Upon fixation, all intracellular trafficking of molecules is stopped. Drying and lipid solvent treatments (acetone, ethanol, etc.) create massive holes in the greater sub/cellular structure. This allows antibodies to cross membranes (extracellular, nuclear, etc.) in fixed cells. There is no detergent that causes an antigen that was previously hidden (masked) to be exposed (unmasked). Low levels of detergents such as Tween 20 in the washing solutions reduce the surface tension and allow the tissues/cells to remain wet.
The most common histological preparative technique is formalin-fixed, paraffin embedded tissue. Some epitopes are more sensitive to fixation and embedding than others and can be masked (hidden) from the addition of affinity reagents. Various antigen retrieval methods exist to unmask a given epitope. These are not applicable to frozen sections. The optimal method will have to determined on a case by case basis. Crosslinking when fixating and embedding can prevent antigen degradation or physical relocation within the cell/tissue. It also eliminates bacterial contamination. Frozen sections can often lose morphological integrity, whereas paraffin embedded sections tend to retain it over multiple sections. Paraffin embedded sections will cause cell/tissue shrinkage that results in higher antigen density over a given section. Always be aware of the fact that your tissues/cells may contain various components that could interfere with your staining technique, such as endogenous enzymes (alkaline phosphatase, peroxidases), endogenous biotin and Fc receptors.
IHC is relatively light-insensitive and allows the visualization of tissue architecture. Autofluorescence can sometimes make IF studies impossible. IF allows for staining of the same subcellular structure with different fluorophores.
NBT/BCIP substrate for AP is the most sensitive but, not widely used because the reaction is slow, does not allow adequate nuclear counterstain, the signal can diffuse and is not compatible with permanent mounting media. Development using DAB substrate for HRP is far more common due to speed of reaction, precise deposition and acceptable color contrast with nuclear stains.
A positive control is needed to monitor the test validity. A negative control is needed to establish a baseline or "zero", to prove that the test is the reason for the measured effect.
WN Buffer Is Used To Remove Protein Residues And Degraded RNA Residues On The Membrane And WS Buffer Is Used To Remove Salt Residues On The Membrane. When A Small Volume Of Bacterial Culture (Less Than 3 Ml) Is Used, The Lysate Resulted Is Usually Not Rich In Protein Contaminants And So Washing With Only WS Buffer Is Already Enough To Result In Plasmid Pure Enough For DNA Sequencing And Other Applications. As One May Notice That When A Product Kit Only Provides One Wash Buffer, It Only Allows Purification Of Plasmid DNA From A Culture Of Volume Less Than 3 Ml. It Is Because One Wash Buffer Is Not Enough To Remove Contaminants From A Higher Volume Of Culture. This Kind Of Product Only Allows Isolation Of High Copy Plasmid As A Small Volume Of Culture Is Used. It Cannot Be Used To Isolate Low Copy Plasmid As A Higher Volume Of Culture Is Required. Moreover, The Drawback Of Using Only One Wash Buffer Is That It Cannot Remove Degraded RNA Bound To The Membrane. Removal Of RNA Existing In The Bacterial Cells Is Achieved By Degrading RNA Released From Cells By RNase Added In MX1 Buffer. Degraded RNA Does Not Bind Well As Undegraded RNA To The Membrane In The Presence Of Chaotropic Salts, Thus Degraded RNA Is Washed Off With Wash Buffer (WN Buffer) Which Contains Chaotropic Salts, Whereas Plasmid DNA Is Still Bound To The Membrane And Is Then Eluted Without RNA Contamination. The Single Wash Buffer Provided In Other Kits Does Not Contain Chaotropic Salts As WN Buffer Does, Thus It Is Not Able To Remove Degraded RNA Bound To The Column. In This Case, Degraded RNA Will Be Co-Eluted With Plasmid DNA. Since RNA Is Degraded, An User Does Not See It By Agarose Gel Electrophoresis Analysis. Though Degraded RNA Does Not Affect Restriction Digestion And Sequencing Reaction, The Presence Of The Ribo-Oligonucleotides Interferes With Some Applications Such As Digestion Of Plasmid With BAL 31 Or Labeling Of The 5’ Termini Of Restiction Enzyme Fragments Of The Plasmid With Bacteriophage T4 Polynucleotide Kinase. Further, The Presence Of Degraded RNA Leads To A False High OD260 Of The Plasmid Eluant (Degraded RNA Also Absorbs Light At Wavelength Of 260 Nm), Thus Misleading The Users That A High Plasmid Yield Is Obtained. The Presence Of Degraded RNA In The Plasmid DNA Solution Can Be Evidenced By OD260/OD280 Ratio Higher Than 1.8. The Use Of Two Wash Buffers Provided In Immunoport's Kit Solves These Problems.
This System Is Mainly Designed To Extract Plasmid DNA From Gram(-) Bacteria Such As E. Coli. Gram(+) Bacteria Have Thicker Cell Wall So Cell Lysis Buffers Provided In The Kit Does Not Lyse Them Readily. However, Extraction Of Plasmid DNA From Gram(+) Bacteria Can Still Be Achieved With Additional Treatment. After Resuspending The Pelleted Bacterial Cells In MX1 Buffer, Add Lysozyme To Give A Final Concentration Of 3 To 5 Mg/Ml. Incubate The Suspension At 37˚C For 30-60 Minutes (Or For A Shorter Time When Use 5 Mg/Ml Lysozyme). This Treatment Weakens The Cell Wall Of The Gram(+) Bacteria. Then Add MX2, And Follow The Rest Of The Protocol. For Certain Gram(+) Bacteria With Thin Cell Wall, Such As Lactobacillus, Applying Of Double Amount Of MX1, MX2, And MX3 Buffer May Have Been Enough To Lyse The Cells. Yet, We Still Recommend Treating Gram(+) Bacteria With Lysozyme To Facilitate Cell Lysis.
When Genomic DNA Is Found In The Eluant, It Means That Genomic DNA Has Been Sheared During Cell Lysis Process. After MX2 Buffer Is Added, Ensure That Mixing Is Done Very Gently To Prevent Genomic DNA Shearing. If Genomic DNA Is Sheared, Genomic DNA Fragments Will Get Into The Lysate And Copurified With Plasmid.
Make Sure That RNase A Is Added Into MX1 Buffer. Store The MX1 Buffer At 4˚C. If RNase A-Added MX1 Buffer Is Not Properly Stored At 4˚C Or Has Been Stored For A Long Time, E.G., More Than 6 Months, RNase A Activity May Have Been Reduced, Thus Not Being Able To Degrade RNA Completely. In This Case, Fresh RNase A Has To Added Into MX1 Buffer With Final Concentration Of 50 Mg/Ml. Again, Store The Buffer At 4˚C.
It Is Possible That Salt Residue In Buffers Or Ethanol Residue In WS Buffer Is Not Removed Completely And Thus Affects The Downstream Reaction. In Case Of Salt Residue, Wash The Column Twice With WS Buffer. In Case Of Ethanol Residue, After Washing With WS Buffer, Make Sure That The Flow-Through Is Discarded And Centrifuge The Column At Full Speed For 3 Minutes. If Necessary, To Centrifuge For A Few Minutes More To Ensure Complete Removal Of Ethanol. Another Reason Is That Plasmid Is Denatured. Denaturation Happens If Incubation In MX2 Buffer Has Gone For Too Long Time. This Can Be Visualized During Electrophoresis That A Band Migrates Faster Than The Supercoiled Form. After MX2 Buffer Is Added, Do NOT Incubate For More Than 5 Minutes.
There Are Several Possible Reasons Accounting For The Lower Plasmid Yield Obtained: 1) Bacterial Culture Did Not Grow Well, Thus Resulting In A Lower Cell Density. To Ensure A Well-Grown Culture, Always Inoculate Bacterial Cells From A Freshly Streaked Plate And Grow Cells In The Presence Of The Required Antibiotic(S). Ensure That Bacteria Have Grown Well After Overnight Culture And Attained An OD600 More Than 1, Meanwhile Do Not Let The Culture Grow More Than 16 Hours, Bacteria May Enter Death Phase And Plasmids In Cells Start To Be Degraded. 2) Do Not Use More Than 5 Ml Of Culture For One Preparation. Sometimes When The Culture Is Too Dense, Cells Collected From A 5 Ml Culture Cannot Be Completely Lysed. Incomplete Cell Lysis Will Lead To A Lower Yield Of Plasmid. 3) If DdH2O Of PH Less Than 7 Is Used For DNA Elution, Lower Efficiency Of Plasmid Elution Will Be Resulted. 4) When A More Concentrated Plasmid DNA Solution Is Desired, 30 Ml Of Elution Buffer Is Suggested. However, In Comparison With Using 50 Ml Elution Buffer, There Is About 40% Of Plasmid Cannot Be Eluted When 30 Ml Is Used. Therefore, No Less Than 30 MlOf Elution Solution Should Be Used. 5) Make Sure That Elution Buffer Is Absorbed Into The Membrane And To Stand The Column For 1-2 Minutes Before Centrifugation To Elute DNA. If The Buffer Still Retains On The Membrane Surface, Plasmid DNA Will Not Be Eluted Due To Lack Of Contact With Buffer. In This Case Pulse Centrifugation Of The Column For 1-2 Seconds (Do NOT Over-Centrifuge) Can Help Permeation Of The Buffer Into The Membrane. 6) Large Plasmid Is Eluted Less Readily Than Small Plasmid. When A Plasmid Is Larger Than 10 Kb, Use Elution Solution Preheated To 70˚C.
It Is Not Recommended To Use Rich Medium Such As TB Or 2X TY For Most Commonly Used Plasmids, Especially High-Copy Number Plasmids. Although Rich Medium Have The Obvious Advantage Of Producing More Bacteria, High Level Of Cell Biomass Reduces The Yield And Quality Of Plasmids. If Rich Medium Must Be Used, Please Reduce The Culture Volume To Match The Suitable Range Of Cell Biomass. If The Culture Used Too Much, Alkaline Lysis Will Be Inefficient To Lower The Yield Of Extracted Plasmids. Furthermore, The Excessive Viscosity Of The Lysate Will Require Vigorous Mixing To Result In Shearing Of Genomic DNA And Subsequent Contamination Of Extracted Plasmids. High Level Of Protein And Polysaccharide Will Usually Be Carried Out With Plasmids To Result In Bad Quality Of Extracted Plasmids. To Incubate Bacterial Cells With Rich Medium Overnight (About 16 Hours) Is Not Recommended. Most Of Antibiotics Will Be Ran Out In 8 Hours. The Culture Will Lose The Selection Of The Antibiotic And Reduce The Yield Of Plasmids In The Bacterial Cells. Incubation With Rich Medium For Long Time, Especially Longer Than 10 Hours, Will Produce Large Amount Of The Dead Cells. Sampling Too Many Dead Cells Will Provide Low Yield And Bad Quality Of Plasmids Extracted.
When Degradation Appears, This Indicates That Nuclease The Possible Presence Of Nuclease In The Eluted Plasmid. There Are Several Things To Do: 1) Nuclease Cannot Be Completely Washed Off Especially When End+ E. Coli Host Strain Is Used. Use End-Strain If Possible. 2) Wash The Column Twice With WF Buffer. 3) Use TE Buffer For Plasmid Elution As EDTA Can Inhibit Nuclease Activity. Store Eluted DNA At -20˚C When Not Used.
The Difference Between Blood & Tissue Genomic DNA Mini And Blood Genomic DNA Midi And Maxi Is That The Midi And Maxi Kits Do Not Have LYS Buffer. LYS Buffer In Blood & Tissue Genomic DNA Mini Is Mainly Used For Sample Digestion Of Tissue Samples. This Means That Any Sample, Which Only Needs EX Buffer For Sample Digestion As Listed In Blood & Tissue Genomic DNA Mini, Can Be Used In The Midi And Maxi Kits. These Samples Include Whole, Buffy Coat, Serum, Plasma, Body Fluid, Lymphocytes, Animal Cells, Bacteria, Viral DNA From Blood Or Body Fluid, And Integrated Viral DNA In Animal Cells. Follow The Blood & Tissue Genomic DNA Mini Protocol For The Procedure But Use The Time Duration And Buffer Volumes As Suggested In That Of Midi And Maxi Kit.
If It Is Blood Stain (Dried Blood) On A Piece Of Filter Paper, It Is Still OK To Use Our Blood & Tissue Genomic DNA Extraction Miniprep System To Extract DNA From It. What Dr. K. Nobuto Can Do Is To Cut The Filter Paper Into Small Pieces (About 10 Mm2). Place 1 To 4 Pieces (Depends On How Concentrated The Blood Stain Is) Into A Clean 1.5-Ml Eppendorf Tube. Add 20 Ml Proteinase K And 200 Ml LYS Buffer Into The Sample. Mix Immediately ByVortexing For 20 Seconds. Follow The Tissue Protocol From Step 3.
Several Points Should Be Noted To Avoid DNA Degradation: 1) DNA Degradation Occurs When The Sample Is Not Fresh Or Is Stored Improperly For A Long Time. Samples Not Used Immediately Should Be Flash Frozen In Liquid Nitrogen And Stored At -80°C. Genomic DNA In Samples Stored At Room Temperature, 4°C, Or -20°C Are Subjected To Degradation. It Is Also Not Advised To Keep Samples In Buffer Or Medium And Stored At -80°C. 2) For Whole Blood Samples, If They Are Stored At Room Temperature For More Than 2 Days Or At 4°C Or -20°C, Genomic DNA Isolated Appears Smearing At An Extent Proportional To The Storage Time. 3) Use Fresh TAE Or TBE Running Buffer For Electrophoresis, Repeatedly Used Running Buffer May Be Contaminated With DNase. 4) If Isolated DNA Needs To Be Stored For A Long Time, Use 10 MM Tris-HCl (PH 9.0) Or TE For Elution. DdH2O Is Not Advised In This Cause Because DNA Fragments In H2O Suffer From Gradual Degradation Through Acid Hydrolysis Readily. 5) If DNA Is To Be Used Frequently, Elute In 10 MM Tris-HCl (PH 9.0) Or TE And Store At 4°C. Keep DNA At -20°C Only For Long-Term Storage. Repeated Freeze-Thaw Cycles Can Cause Shearing Of Genomic DNA. 6) Genomic DNA Extracted From Paraffin-Embedded Tissue Is Usually Degraded. It Is Because Genomic DNA In Paraffin-Embedded Tissue Unavoidably Suffers From Degradation When Sample Was Treated And Stored For A Long Time. DNA In This Case Is Not Suitable For Southern Blotting And Restriction Analysis Due To The Smearing. However, It Is Applicable For PCR.
This Indicates That RBC Or Hemoglobins Have Not Been Completely Lysed Or Digested. During Incubation For Proteinase K Digestion, The Sample Should Be Mixed Every 3-5 Minutes By Vortexing And Inverting To Completely Disperse Proteinase K And Samples.
The Key Is To Use Fresh Sample And Not To Overload The Column. Low Yield Or Purity Of Genomic DNA Is Usually Due To Incomplete Digestion Or Incomplete Lysis Of The Sample. Starting With A Maximum Amount Or Volume Of Samples Does NOT Usually Give The Best Yield Of DNA. On The Contrary, It Usually Results In Incomplete Sample Lysis And Degradation Of Proteins, Thus Making Extraction Of All DNA From The Sample Unfeasible. Further, It Always Requires Subsequent Removal Of Undigested Residues And Yields Viscous Sample Lysate. When The Lysate Is Too Viscous, It Not Only Has Difficulty In Passing The Column, But Also Indicates The Presence Of An Abundant Amount Of Contaminants Such As Proteins And Salts. Contaminants Of High Amount Not Only Affect DNA Binding, But Also May Not Be Washed Off Completely, Leading To Carry Over To The Eluted Genomic DNA. Therefore, A Good Quality And Yield Of DNA Is Only Expected When A Sample Is Completely Digested. We Advise Starting With Half Of The Maximum Amount Of Sample Suggested. When There Is No Problem In Digesting The Sample Completely And Passing The Lysate Through The Column, Amount Of The Sample To Be Applied Can Be Increased Gradually In The Subsequent Preparations.
If A Sample Is Rich In Protein, E.G. Fish Flesh, Complete Digestion Will Not Be Achieved Using The Amount Of Proteinase K And Buffer Suggested In The Protocol. If A Sample Cannot Be Digested Completely Or Appears Very Viscous, Add More LYS Buffer And Repeat Incubation. Centrifuge The Sample At Full Speed For 5 Minutes To Remove Undigested Remains And Only Use The Supernatant In The Following Steps. In The Subsequent Preparations, A Lower Amount Of The Sample Should Be Used. A General Rule Of Thumb Is To Start With Half Of The Maximum Amount Of Sample Suggested. When There Is No Problem In Digesting The Sample Completely And Passing The Lysate Through The Column, Amount Of The Sample To Be Applied Can Be Increased Gradually In The Next Preparations.
Genomic DNA Extracted From Paraffin-Embedded Tissue Is Usually Degraded. It Does Not Show As A Clear Strong Band Of About 20-30 Kb As We Usually See When Fresh Sample Is Used. It Is Because Genomic DNA In Paraffin-Embedded Tissue Unavoidably Suffers From Degradation When Sample Has Been Treated And Stored Over A Long Period. DNA In This Case Is Not Suitable For Southern Blotting And Restriction Analysis Due To The Smearing. However, It Is Applicable For PCR.
or Yeast, Use Blood & Tissue Genomic DNA Extraction System. For Filamentous Fungi And Mushroom, Use Plant Genomic DNA Extraction System. Yield of genomic DNA extracted is low. Several Things To Pay Attention To: 1) The Sample May Not Contain Enough Amount Of Genomic DNA. Increase The Sample Amount, Proteinase K, And Buffers Proportionally. 2) Elution Buffer Preheated At 70°C Is Used. 3) DdH2O, Tris-HCl, Or TE Used Should Be Of PH 8.0-9.0. 4) After Adding Elution Buffer Into The Column, Stand The Column For 1-5 Minutes Before Centrifuging To Collect Eluted DNA.
This Indicates That The Number Of WBC (White Blood Cell) In The Buffy Coat Is Too High, Thus Not Being Lysed And Digested Completely By Proteinase K. Buffy Coat Should Be Prepared From A Lower Volume Of Whole Blood And To Make Sure That Fewer Than 1x107 Of WBC Is Used Per Preparation. Incubation Should Be Done With Constant Mixing To Disperse Proteinase K And Sample. If Lysis Is Incomplete, Add More Proteinase K And Repeat Incubation. The Sample Should Not Contain Insoluble Residues When It Is Completely Digested. Centrifuge To Remove Any Undigested Residues And Use Only The Supernatant To Continue The Procedure.
Yes, PX Buffer Does Not Affect The Chemically Linked DIG On DNTP. Similarly, This System Can Be Used To Clean Up 32P-Labeled DNA Fragment.
Both Systems Can Be Used To Clean Up DNA Fragments (100-Bp To 10-Kb) From Enzymes, Salts, And DNTPs. PCR AdvancedTM Clean Up System Is A Cheaper Choice For Cleaning Up DNA Fragments In Solution. When A Specific DNA Fragment Is To Be Purified, Gel Electrophoresis Is Needed. A Gel Slice Containing The Desired DNA Fragment Is Excised, And DNA Is Extracted Using Gel AdvancedTM Gel Extraction System.
It Is Not Recommended To Use This System To Clean Up Sequencing Reaction Because PCR Products Smaller Than 100-Bp Cannot Be Recovered Effectively, Thus Making Reading Of The First 80-100 Nucleotide Sequence Unfeasible.