Protocol. Quantitative Western Blot Analysis with Replicate Samples

Transcription

1 Prtcl Quantitative Western Blt Analysis with Replicate Samples Published June Revised December The mst recent versin f this Technical Nte is psted at licr.cm/bi/supprt.

2 Page 2 - Quantitative Western Blt Analysis with Replicate Samples Table f Cntents Page I. Intrductin 2 Types f Replicate Measurements 3 Reprting Changes in Relative Abundance 3 II. Keys t Success 5 III. Experimental Design 6 Analysis f Replicate Samples n Separate Western Blts 6 IV. Quantitative Western Blt Nrmalizatin 7 V. Calculatins fr Replicate Analysis 8 VI. Data Interpretatin 11 VII. References 14 VIII. Further Reading 15 I. Intrductin Replicate measurements are critical fr quantitative Western blt (QWB) analysis. The purpse f QWB is t mnitr changes in the relative abundance r mdificatin f a target prtein within a grup f samples. Des the experimental treatment cause an increase r decrease in target abundance, cmpared t the untreated r cntrl cnditin? Replicate samples cnfirm the validity f bserved changes in prtein levels. Withut replicatin, it is impssible t knw if an effect is real r simply an artifact f experimental nise r variatin. Bilgical and technical replicates are bth imprtant, but each type f replicate addresses different questins (1, 2, 3).

3 Quantitative Western Blt Analysis with Replicate Samples - Page 3 Types f Replicate Measurements Technical replicates are repeated measurements used t establish the variability f a prtcl r assay, and determine if an experimental effect is large enugh t be reliably distinguished frm the assay nise (1). Examples may include lading f multiple lanes with each sample n the same blt, running multiple blts in parallel, r repeating the blt with the same samples n different days. Technical replicates evaluate the precisin and reprducibility f an assay, t determine if the bserved effect can be reliably measured. When technical replicates are highly variable, it is mre difficult t separate the bserved effect frm the assay variatin. Yu may need t identify and reduce surces f errr in yur prtcl t increase the precisin f yur assay. Technical replicates d nt address the bilgical relevance f the results. Bilgical replicates are parallel measurements f bilgically distinct and independently generated samples, used t cntrl fr bilgical variatin and determine if the experimental effect is bilgically relevant. The effect shuld be reprducibly bserved in independent bilgical samples. Demnstratin f a similar effect in anther bilgical cntext r system can prvide further cnfirmatin. Examples include analysis f samples frm multiple mice rather than a single muse, r frm multiple batches f independently cultured and treated cells. T demnstrate the same effect in a different experimental cntext, the experiment might be repeated in multiple cell lines, in related cell types r tissues, r with ther bilgical systems. An apprpriate replicatin strategy shuld be develped fr each experimental cntext. Several recent papers discuss cnsideratins fr chsing technical and bilgical replicates (1, 2, 3). Reprting Changes in Relative Abundance QWB methds typically use ratimetric analysis t determine relative abundance f the target prtein and cmpare relative prtein levels acrss a grup f samples. Ratimetric analysis is a frm f "self-calibratin" that expresses the relative abundance f target prtein as a rati between the experimental sample and the cntrl. It cmpares the intensity f the target band in each experimental sample t the intensity f the target band in the cntrl sample (4, 5, 6). Nrmalize yur QWB data using an apprpriate internal lading cntrl (such as ttal prtein staining).

4 Page 4 - Quantitative Western Blt Analysis with Replicate Samples Nrmalizatin mathematically crrects fr small, unavidable variatins in sample lading and transfer by cmparing the target prtein t an internal lading cntrl. Mre infrmatin is fund in Sectin IV. See these resurces t learn mre abut QWB nrmalizatin: Western Blt Nrmalizatin Handbk (licr.cm/handbk) Western Blt Nrmalizatin: Challenges and Cnsideratins White Paper (licr.cm/nrmalizatinreview) Perfrm the ratimetric analysis, as described in Sectin V. The nrmalized signal intensity f the target band in each sample shuld be divided by the nrmalized intensity f the target band in the cntrl sample. The resulting ratis, expressed as fld change r percentage (%) change, are used t cmpare relative prtein levels acrss the samples n yur blt. Because all samples are cmpared t the cntrl, these measurements are prprtinal and are independent f raw signal intensity. Fld change is a unitless value that cmpares the relative abundance f a target prtein t the cntrl sample n the same membrane. A value abve 1.0 indicates an increase in abundance relative t the cntrl; a value belw 1.0 indicates decreased abundance (Table 1). Percentage (%) change is a unitless value similar t fld change that expresses changes in relative abundance as a percentage. A psitive percentage indicates increased abundance relative t the cntrl; a negative percentage indicates decreased abundance (Table 1).

5 Quantitative Western Blt Analysis with Replicate Samples - Page 5 Table 1. Relative abundance f target prtein, expressed as fld change and % change. Fld Change % Change 1.0 0% % % % % % % II. Keys t Success Accurate QWB analysis requires a rbust, well-characterized detectin system. Data shuld be captured within the linear range f detectin, with careful attentin t limits f detectin and saturatin f strng bands. Methds shuld be sufficiently rbust that the utcme is unaffected by trivial r unintended changes in the reagents and prtcl (7-8). Mre Inf: This prtcl prvides guidelines fr characterizing the linear range f detectin fr yur system: Determining the Linear Range fr Quantitative Western Blt Detectin (licr.cm/linearrange) Relative changes in abundance can nly be measured if they substantially exceed the variability f yur measurement. See Sectin VI, Data Interpretatin, fr guidelines. D nt cmpare signal intensities between blts. Experimental variatins in sample lading transfer cnditins, antibdy reagents, incubatin times, and many ther factrs affect the raw signal intensities n each blt. Band intensity shuld nly be analyzed by relative, ratimetric cmparisn t the cntrl samples. Every blt shuld include cntrl samples (untreated, baseline, etc.) and be prcessed fr detectin with the same reagents, prtcl, and imaging methd. Fld change values can then be cmpared between blts laded with the same cntrl samples.

6 Page 6 - Quantitative Western Blt Analysis with Replicate Samples III. Experimental Design Analysis f Replicate Samples n Separate Western Blts 1. Plan an apprpriate strategy fr technical and bilgical replicates in yur experiment. It may be helpful t cnsult a statistician befre yu begin t generate data. 2. Cllect replicate data that represent the desired experimental cnditins (drug treatments, time curse, dse-respnse, etc). A minimum f three replicates shuld be perfrmed fr each sample, including the cntrls. Imprtant: Samples shuld be laded in randm rder n each blt t minimize psitin effects. Sample placement n the blt (fr example, in edge lanes vs. interir lanes) may affect signal intensity and intrduce variability. Randm sample placement will reduce the impact f psitin effects (4). Figure 1. Replicate blt layut described in this prtcl. Each gel is laded with MW marker (M), cntrl sample (C), and experimental samples (1-10). On blts B and C, cntrl and experimental samples are laded in randm rder. 3. Use this prtcl t analyze and cmpare replicate samples n separate Western blts. a. This prtcl describes the analysis f three replicate blts (designated A, B, and C; see Figure 1), each laded with a mlecular weight marker, cntrl sample, and 10 experimental samples. b. This layut can be used t analyze either technical r bilgical replicates, depending n the nature f the samples yu lad. Apprpriate interpretatin f the resulting data will be specific t yur samples and experimental cntext. Technical replicates: Lad the same grup f cntrl and experimental samples repeatedly, nt three (r mre) separate gels, t generate replicate blts. Bilgical replicates: Perfrm three (r mre) independent experiments. Lad each set f cntrl and experimental samples nt its wn separate gel, t generate replicate blts.

7 Quantitative Western Blt Analysis with Replicate Samples - Page 7 IV. Quantitative Western Blt Nrmalizatin 1. Nrmalize yur Western blt data, using an apprpriate internal lading cntrl. a. Nrmalizatin mathematically crrects fr small, unavidable sample-t-sample and lanet-lane variatin by cmparing the target prtein t an internal lading cntrl. Internal lading cntrls are endgenus reference prtein(s) that are present in all samples at a stable level, and unaffected by the experimental cnditins r treatments. The lading cntrl serves as an indicatr f sample prtein lading, and is used t verify that bserved changes in target prtein abundance represent actual differences in the prtein samples. b. Cmmn internal lading cntrls include ttal prtein staining, husekeeping prteins (such as actin, tubulin, r GAPDH), and analysis f prtein mdificatins with pan- and mdificatin-specific primary antibdies. Ttal prtein staining: The membrane is treated with a ttal prtein stain t assess sample prtein lading in each lane. After immunbltting, the cmbined signal f all sample prteins in each lane is used as a lading indicatr fr nrmalizatin. This methd is recmmended by the Jurnal f Bilgical Chemistry (9). Prtcl: REVERT Ttal Prtein Stain Nrmalizatin (licr.cm/revertnrmalizatin) Husekeeping prtein (HKP): This ppular methd emplys a single, unrelated endgenus prtein as a readut f sample lading. Because a single indicatr is used, changes in HKP expressin will intrduce errr. Validatin is required t verify that HKP expressin is cnstant in all samples and unaffected by experimental cnditins. Prtcl: Husekeeping Prtein (HKP) Validatin (licr.cm/hkp-validatin) Prtcl: Husekeeping Prtein Nrmalizatin (licr.cm/hkp-nrmalizatin) Pan- and mdificatin-specific antibdies: This specialized frm f nrmalizatin is used t analyze prtein phsphrylatin and ther pst-translatinal mdificatins. A mdificatin-specific primary antibdy is multiplexed with a pan-specific antibdy that recgnizes the unmdified target prtein. Signals are nrmalized t the actual level f target in each lane, using the target prtein as its wn internal cntrl. Prtcl: Pan/Phsph Analysis fr Western Blt Nrmalizatin (licr.cm/panprteinnrmalizatin) 2. Use the nrmalized band intensity values fr analysis f technical r bilgical replicates, as in Sectin V.

8 Page 8 - Quantitative Western Blt Analysis with Replicate Samples V. Calculatins fr Replicate Analysis 1. Prepare an analysis spreadsheet with the nrmalized band intensity values fr each replicate (as described in Sectin IV). Nte: Western blt data must be nrmalized BEFORE replicate analysis is perfrmed. See Sectin IV fr mre infrmatin. 2. Using the nrmalized values, calculate the fld change (rati f the experimental sample t the cntrl) fr each sample n replicate blt A (depicted in Figure 2). BLOT A Sample Target, nrm. intensity Cntrl, nrm. intensity Fld change C 10,000 10, ,000 10, ,000 10, ,000 10, ,000 10, ,000 10, ,000 10, ,000 10, ,000 10, ,000 10, ,000 10, Figure 2. Fld change calculatins: example data fr Blt A. (left) In the blt diagram, C indicates the untreated cntrl sample. Samples 1-10 received the experimental treatments. M indicates mlecular weight marker. (right) Fld change was calculated fr each band, using the nrmalized intensity values frm Blt A. Each experimental sample is cmpared t the cntrl, t generate a rati. Example values are shwn fr illustratin.

9 Quantitative Western Blt Analysis with Replicate Samples - Page 9 3. Repeat the fld change calculatins with the nrmalized values frm replicate blts B and C (depicted in Figure 3). Sample Blt A fld change Blt B fld change Blt C fld change C Figure 3. Fld change calculatins. Example data are shwn fr Blts A, B, and C. (tp) Strategy fr calculating the fld change. (bttm) Fld change values fr the three blts. In this illustratin, samples 1-10 were laded n replicate blts (Blts A, B, and C are technical replicates). 4. Using the fld change values frm each replicate blt, calculate the mean fld change, verall % change, standard deviatin, and cefficient f variatin (CV) fr all replicates (Table 2). a. Calculate the mean fld change f each replicate measurement. b. Calculate the standard deviatin f the fld change fr each replicate measurement.

10 Page 10 - Quantitative Western Blt Analysis with Replicate Samples c. Calculate the cefficient f variatin(cv) f the fld change fr each replicate measurement. Table 2. Mean, % change, standard deviatin, and CV calculatins. Example data are shwn fr analysis f samples 1-10 n Blts A, B, and C ( n = 3 technical replicates). Sample Blt A fld change Blt B fld change Blt C fld change Mean fld change % change StDev CV C % % % % % % % % % % % % % % % % % % % % % % 5. Plt the fld change in prtein expressin as a functin f the treatment (Figure 4). a. Sme jurnals nw prefer that small datasets be presented in scatterplt frmat, rather than the mre typical bar graphs (9-10). Scatterplts clearly shw the spread and distributin f yur data pints, making it easier fr peer reviewers and readers t fully understand yur results. Bar graphs are mre suitable fr large datasets. Many jurnals nw discurage the use f errr bars that indicate the standard errr f measurement (SEM). Errr bars that indicate the standard deviatin f yur measurements may be mre apprpriate (11, 12, 13). Figure legends shuld clearly state what the errr bars n the graph represent, and the value f n fr the experiment. Sufficient detail shuld be prvided t indicate if replicates are technical r bilgical.

11 Quantitative Western Blt Analysis with Replicate Samples - Page 11 Figure 4. Bar graph and scatterplt f the fld change data in Table II. (left) Mean fld change was pltted fr each sample n a bar graph. Errr bars indicate the standard deviatin fr each measurement (n = 3). The cntrl is indicated by a black bar. (right) Fld change values frm all blts were displayed as a scatterplt. The mean fld change fr each sample is indicated by a hrizntal line. The cntrl sample is represented by a black dt. Technical replicates f samples 1-10 are shwn (n = 3). VI. Data Interpretatin Imprtant: General guidelines are presented in this prtcl. Apprpriate replicatin and data interpretatin are specific t each experiment, and beynd the scpe f this prtcl. Cnsult yur lcal statistician fr assistance. 1. Use the fld change and % change values fr relative cmparisn f samples. 2. % change and CV can be used t evaluate the rbustness f yur QWB results, and determine if the magnitude f bserved changes is large enugh t be reliably distinguished frm assay variability. a. As described in Sectin II, % change expresses the fld change as a percentage. A psitive number indicates an increase in relative abundance f target prtein. A negative number indicates decreased abundance. b. The cefficient f variatin(cv) describes the spread r variability f measured signals by expressing the standard deviatin (SD) as a percent f the mean. Because CV is independent f the mean and has n unit f measure, it can be used t cmpare the variability f data sets and indicate the precisin and reprducibility f an assay.

12 Page 12 - Quantitative Western Blt Analysis with Replicate Samples A lw CV value indicates lw signal variability and high precisin f measurement. A larger CV indicates greater variatin in signal and reduced precisin 3. On a Western blt, a change in band intensity is meaningful nly if the magnitude f the % change substantially exceeds the CV (Table 3). a. Generally speaking, the magnitude f the reprted % change shuld be at least 2X greater than the CV f that measurement. Fr example, t reprt a 20% difference between samples (0.8-fld r 1.2-fld change in band intensity), CV < 10% wuld be recmmended fr replicate samples. Fr a specific measurement, this threshld fr the magnitude f change wuld crrespnd t the mean ± 2 SD. In Table 3, the change bserved in sample 2 is nt meaningful (grey shading). A small change in target abundance was bserved (-3%), but the CV f the measurement (22%) was larger than the effect. Samples 8, 9, and 10 (range shading) each shwed a mderate increase in target abundance. In each case, the reprted % change was cnsiderably greater (>2.5X) than the CV f the fld-change measurement. Table 3. CV and reprted % change. The bserved change in sample 2 (grey) is smaller than the CV f the measurement, and is nt meaningful. In samples 8, 9, and 10 (range), the reprted change exceeds the CV by at least 2.5X. These changes are likely t be significant. Technical replicates f samples 1-10 are shwn (n = 3). Sample Blt A Blt B Blt C Mean % Change StDev CV fld change fld change fld change fld change C % % % % % % % % % % % % % % % % % % % % % % b. Faint bands r subtle changes in band intensity are mre difficult t detect reliably. In these situatins, QWB analysis requires mre extensive replicatin and a lwer CV.

13 Quantitative Western Blt Analysis with Replicate Samples - Page 13 Technical replicates: If the CV f yur technical replicates is high, yu may need t identify and reduce surces f errr in yur prtcl t increase the precisin f yur assay. Technical replicates help yu characterize the variability f yur assay, s yu can determine if the experimental respnse is reliable and meaningful. Bilgical replicates: Interpret yur technical replicatin data in the cntext f yur bilgical replicates. A strng experimental respnse may be easily demnstrated, even with substantial technical variatin. Hwever, a weak r variable respnse may be very difficult t discriminate frm the assay nise, and may require mre extensive technical replicatin (1). Nte: These are general guidelines nly. Replicatin needs and data interpretatin are specific t yur experiment, and yu may wish t cnsult a statistician.

14 Page 14 - Quantitative Western Blt Analysis with Replicate Samples VII. References 1. Naegle K, Gugh NR, Yaffe MB. Criteria fr bilgical reprducibility: what des n mean? Sci Signal. 8 (371): fs7 (2015). 2. Blainey P, Krzywinski M, Altman N. Replicatin: quality is ften mre imprtant than quantity. Nat Meth. 11(9): (2014). 3. Vaux DL, Fidler F, Cumming G. Replicates and repeats what is the difference and is it significant? EMBO reprts 13(4): (2012). 4. Aldridge GM, Pdrebarac DM, Greenugh WT, Weiler IJ. The use f ttal prtein stains as lading cntrls: an alternative t high-abundance single prtein cntrls in semi-quantitative immunbltting. J Neursci Meth. 172(2): (2008). 5. McDnugh AA, Veiras LC, Minas JN, Ralph DL. Cnsideratins when quantitating prtein abundance by immunblt. Am J Physil Cell Physil. 308: C426-C433 (2015). 6. Bakkenist CJ, Czambel RK, Hershberger PA, Tawbi H, Beumer JH, Schmitz JC. A quasi-quantitative dual multiplexed immunblt methd t simultaneusly analysis ATM and H2AX phsphrylatin in human peripheral bld mnnuclear cells. Oncscience 2(5): (2015). 7. Plant AL, Lcasci LE, May WE, Gallagher PD. Imprved reprducibility by assuring cnfidence in measurements in bimedical research. Nat Meth. 11(9): (2014). 8. Janes KA. An analysis f critical factrs fr quantitative immunbltting. Sci Signal 8(371): rs2 (2015). 9. Fsang AJ, Clbran RJ. Transparency is the key t quality. J Bil Chem. 290: (2015). 10. Editrial. Twards greater reprducibility fr life-sciences research in Nature. Nature 546: 8 (2017). 11. Krzywinski M, Altman N. Errr bars. Nat Meth. 10(10): (2013). 12. Fay DS, Gerw K. A bilgist s guide t statistical thinking and analysis. WrmBk, ed. The C. elegans Research Cmmunity, WrmBk, di/10.189/wrmbk , (2013). 13. Nagele P. Misuse f standard errr f the mean (SEM) when reprting variability f a sample. A critical evaluatin f fur anaesthesia jurnals. Br J Anaesth. 90:

15 Quantitative Western Blt Analysis with Replicate Samples - Page 15 VIII. Further Reading Please see the fllwing fr mre infrmatin abut QWB analysis. Western Blt Nrmalizatin Handbk licr.cm/handbk The Nrmalizatin Handbk describes hw t chse and validate an apprpriate internal lading cntrl fr nrmalizatin. Gd Nrmalizatin Gne Bad licr.cm/gngb Gd Nrmalizatin Gne Bad presents examples f nrmalizatin that have been adversely affected by cmmn pitfalls and ffers ptential slutins. Western Blt Nrmalizatin White Paper licr.cm/nrmalizatinreview This white paper cmprehensively reviews the literature f Western blt nrmalizatin. Determining the Linear Range fr Quantitative Western Blt Detectin licr.cm/linearrange This prtcl explains hw t chse an apprpriate amunt f sample t lad fr QWB analysis. REVERT Ttal Prtein Stain Nrmalizatin Prtcl licr.cm/revertnrmalizatin This prtcl describes hw t use REVERT Ttal Prtein Stain fr Western blt nrmalizatin and analysis. Pan/Phsph Analysis Fr Western Blt Nrmalizatin licr.cm/panprteinnrmalizatin This prtcl describes hw t use pan-specific antibdies as an internal lading cntrl fr nrmalizatin. Husekeeping Prtein Validatin Prtcl licr.cm/hkp-validatin This prtcl explains hw t validate an HKP fr use as an internal lading cntrl, by demnstrating that HKP expressin is stable in the relevant experimental samples. Husekeeping Prtein Nrmalizatin Prtcl licr.cm/hkp-nrmalizatin This prtcl describes hw t use a husekeeping prtein fr Western blt nrmalizatin and quantitative analysis.

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