ponceau staining protocol pvdfclubman pinaud cologne

Quantification of 4g and 10g of protein stained with 0.1% (w/v) Ponceau S with either 1% (v/v) acetic acid (AA), 5% (v/v) AA, or 3% (v/v) TCA for 2 minutes. We suggest the use of the relatively inexpensive 0.01% Ponceau S in 1% acetic acid stain for total protein normalization as it is as effective as all the expensive formulations that are currently used. The staining intensity of solutions containing TCA was similar to when acetic acid was used (Figure 5). Band intensities were totaled for each lane. To determine the effect of Ponceau S concentration on the detection of proteins, nitrocellulose membrane containing different concentrations of cytosolic liver lysates were stained with different Ponceau S concentrations (0.1-0.5% w/v) in 1% acetic acid for 2 minutes each (Figure 2). OwR%?6Yi1,L~v7-c0k;b7m7_dU i@lH2TOD;UJdDgdqHF^q>N?0+QyG=|GKOq|_v? Western blots are usually carried out with total protein loading amounts in the range of 10-20g. 0000000016 00000 n will also be available for a limited time. stream tNdpM|}b.u"wSNm rxVSr@P J.5 ~gCaTWCUYK\M9iE8O{!58[PfzOD[&q5ZV&(,Sg{Zf)Kl|Kp7hVBpv`9 )lY:\qM0Qy8 F@26na Qy8X$ ,Vi1qU Q#TK/$Eokn&{=(IA!?X9rHez WK,"z>M)K,Z~ };PpJ4 V/Z]Tu `^3CiD# y=a#e In experiments to investigate if a combination of TCA and sulfosalicylic acid could enhance the sensitivity of detection of protein bands, staining with 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid was compared to 1% (v/v) acetic acid (AA), 0.02% (w/v) Ponceau S with 0.3% (v/v) TCA and 0.3% (w/v) sulfosalicylic acid, and 0.2% (w/v) Ponceau S with 3% (v/v) TCA and 3% (v/v) sulfosalicylic acid (Figure 7). In the last decade several publications have suggested that the commonly used housekeeping proteins are not expressed at the same levels across cell types and experimental conditions [2; 4; 5; 6; 7; 8]. The staining is reversible and compatible with subsequent Western blotting [, Incubate membrane in Ponceau S solution for 5-10 minutes at room temperature (RT). Another less commonly used method for total protein normalization is staining the membrane prior to immunodetection with a total protein stain to determine sample loading in each lane. C) Western blots of membranes stained with 0.01% Ponceau S in 5% acetic acid, 2% Ponceau S in 30% SA and membranes not stained with Ponceau S. Western blots were carried out as described in the methods using anti-PSMA3 antibody. In this report we investigated the different Ponceau S formulations currently utilized as well as others not currently utilized to determine the optimal Ponceau S stain for Western blot normalization. Ponceau S is the most commonly used stain for total protein normalization. Ponceau S is likely to be one of the most commonly used stains for normalizing Western Blots in the near future. aDepartment of Neurobiology, Physiology, and Behavior, University of California, Davis, CA, US, bDepartment of Physiology and Membrane Biology, University of California, Davis, CA, US. The lower blots show the same blots destained and then restained with commercial Ponceau S and the lower bar charts show the normalized results. 0000011990 00000 n To effectively determine the relative amount of a target protein on a blot, a normalization control is needed. Membranes were then washed 3 times for 5 minutes each with TBST. 0000014020 00000 n Second, the housekeeping gene and protein levels can change during developmental phases and under different experimental conditions. The lower blots show the same blots destained and then restained with commercial Ponceau S and the lower bar charts show the normalized results. The dye rapidly stains proteins on membranes pink or light red for easy visual inspection of protein transfer. Quantification of 4g and 10g of protein stained with 1% (v/v) acetic acid and either 0.1%, 0.2%, or 0.5% (w/v) Ponceau S for 2 minutes. To prepare the strips for Western blotting, the strips were once again destained with TBST. Different concentrations of acetic acid have been recommended [16; 17]; therefore, to determine if acetic acid concentrations had any effect on Ponceau S staining intensity, 0.1% (w/v) Ponceau S in 1%, 10%, and 20% (v/v) acetic acid were investigated (Figure 4). hb```f``g`c` B,@Q&?%%yL86aXg40,G2k4egc Z0Q9GDBa/PUV\40,l`Zpm The lower bar charts show the results obtained (normalized results) when the commercial Ponceau S staining was used to normalize the upper blots containing different Ponceau S compositions. The averages and standard deviations were found for each set of experiments. Liver from 3-month-old male and female rats were pooled and homogenized in a homogenization buffer containing 50mM Tris, 1mM EDTA, 150mM NaCl, 5mM MgCl2, pH 7.4 using a Dounce homogenizer and centrifuged at 12000 rpm for 15 minutes at 4C. Non-validated antibodies often result in artifactual results [3]. :(zu 0000014659 00000 n Wash the membrane in distilled water (DI) for 1-5 minutes until pink protein bands are visible. The upper blots show the effect of different Ponceau S concentrations on the relative amount of protein detected, while the lower blots show the same blots destained and then restained with commercial Ponceau S. The lower bar charts show the different Ponceau S concentrations normalized to the commercial Ponceau S stain. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. :v= 0;GOT &*8s3u@ Y+LoXCJkf*kJ!F-In:1y(B( +K\ Image membrane and then wash in 1X TBST multiple times for 5 minutes at RT until protein bands are no longer visible [, Transfer membrane to 5ml Ponceau S stain solution on an orbital shaker for 5 minutes at RT. After trying several additives including polyvinylpyrrolidone (PVP) and glycerol, we found no significant increase in the sensitivity of the Ponceau S stains (data not shown). Surprisingly, the lowest concentration of Ponceau S commonly used in the literature (0.1% Ponceau S) showed similar protein detection sensitivity as 0.01 and 0.001% (w/v) Ponceau S stains suggesting that Ponceau S is currently being used at significantly higher concentrations than needed. Quantification of 4g and 10g of protein stained with 0.1% (w/v) Ponceau S with 1%, 10%, and 20% (v/v) acetic acid for 2 minutes. 0000001321 00000 n 0000037639 00000 n The Ponceau S acid solutions used included 0.0001, 0.001, 0.01, 0.1, 0.2, 0.5% (w/v) Ponceau S in 1%, 5% (v/v) acetic acid, 3%, 30% (w/v) TCA or 30% (w/v) sulfosalicylic Acid. 0000003398 00000 n The most commonly used concentration of Ponceau S is 0.1%, while 0.001% (100-fold less) Ponceau S resulted in the same sensitivity of protein band detection. %PDF-1.3 Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, and Heinen E, Housekeeping genes as internal standards: use and limits, Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Effect of sulfosalicylic acid on Ponceau S protein staining intensity. << /Length 1 0 R /Filter /FlateDecode >> Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. FOIA Stain-Free total protein staining is a superior loading control to beta-actin for Western blots, Guidelines for authors and reviewers on antibody use in physiology studies, American journal of physiology. 41 0 obj <> endobj xref The membrane strips were blocked at room temperature using 3% non-fat milk (Bio-Rad) in TBST for 1 hour on a shaker set at 100 rpms. For protein destaining, wash the membrane with 0.1N NaOH solution for 5 minutes and repeat once. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. 0000001384 00000 n This is an antibody-independent method that utilizes the collective signal from the many proteins in one sample lane, as opposed to the use of a single protein when a housekeeping protein is utilized. }Zt2-l5=EbkOG UEEuAr-wK,3D 0000037392 00000 n Currently, a wide variety of Ponceau S formulations have been used to assess the quality of a transfer, locate proteins, and carry out normalization. $3mJzOwwX?F-qdzfeV-G|$A!oy=zF Q -UjvIv ! Before 0000011925 00000 n Ponceau S solutions with 3% (v/v) TCA have been utilized in the literature [11]. The methods recommended to stain and destain membranes with Ponceau S stain is also variable (Table 1). Electrophoresis was carried out at 120V for 70 minutes or until dye front reached the bottom of the gel. The lower percentage of Ponceau S staining solutions are better for the environment and for general safety. The most commonly used normalization control is housekeeping protein normalization. sdj>@eg=\2pgb)FH\R%Q2+ 0000006499 00000 n Lanes were manually fit to each lane and all bands including faint bands were detected. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. Rat livers were obtained from Pel-Freez Biologicals (Arkansas), Nitrocellulose (Trans-Blot Turbo Midi Nitrocellulose Transfer Packs #1704159, Bio-Rad). 0000006891 00000 n For chemiluminescent imaging, Clarity ECL Substrate (Bio-Rad) was used to detect the HRP-conjugated secondary antibody. We were unable to find an optimal Ponceau S stain as all the stains worked equivalently. )K]aVn#^15K7{U /.Mf`DEU&bZ. x]Ms7We"vs%yW3G\(;dS"EyB5d:llY /_f~}}KW>qd]=#N.T6tKz0? Housekeeping proteins can also be probed after stripping a blot that was previously used to detect the target protein. Errors can arise from many sources including sample preparation, unequal sample loading, or uneven transfer, such as when bubbles prevent protein transfer from the gel to the membrane [1; 2; 3]. Simply transfer the membrane to a preferred blocking buffer which will automatically de-stain the membrane. Quantification of 4g and 10g of protein stained with 3% (v/v) TCA with 0.1%, 0.2%, and 0.5% (w/v) Ponceau S for 2 minutes. The lower blots show the same blots destained and then restained with commercial Ponceau S and the lower bar charts show the normalized results. Ponceau S, or 3-Hydroxy-4-(2-sulfo-4-[4-sulfophenylazo]phenylazo)-2,7-naphthalenedisulfonic acid sodium salt, is the most commonly used reversible stain put on membranes before Western blotting [11; 12]. 0000005321 00000 n Ponceau S, a total protein stain, was recently recommended by the Journal of Biological Chemistry and the American Journal of Physiology [9; 15] as the preferred method for normalization of Western blots. !tP.[3 jFg;9j/Nxwn)J0-dX.bk3%E`9~(S 0000038460 00000 n The most commonly used formulations utilize between 0.1% and 2% Ponceau S. The solvent for the typical Ponceau S stain is also highly variable, ranging from 1% acetic acid to 30% trichloroacetic acid and 30% sulfosalicylic acid. Following gel electrophoresis and transfer to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane, this diazo dye can be applied to reveal protein bands with its red/pink stain while leaving a clear background [14]. 0000013436 00000 n Careers, The publisher's final edited version of this article is available at, Total protein staining offers several advantages for Western blotting normalization, Ponceau S is the most common total protein stain for Western blotting normalization, Most laboratories are using excessive amounts of Ponceau S and different acids, 0.01% (w/v) Ponceau S in 1% acetic acid is as effective as all other formulations, Only 1-2 minutes of staining is required for total protein staining with Ponceau S, Ponceau S, Total protein staining, Western blot normalization, {"type":"entrez-nucleotide","attrs":{"text":"LC255201","term_id":"1345669231","term_text":"LC255201"}}, The necessity of and strategies for improving confidence in the accuracy of western blots, Protein purification and analysis: next generation Western blotting techniques. Several total protein stains are available including Ponceau S and Stains Free Stains [8; 9; 10]. ?Q'v?vr29ozcCEev0q; !zG`@%"(b\Qp=zzDkznGz|f #p='Fz p=GXcczf=z\c=z\g~B\Rz~BA0_ 6i zd=zb.QA&K;3_oWntsT: Staining is reversible by washing the membrane with 0.1 M NaOH for 1 min [, A rapid, reversible stain for the detection of proteins in Western blot experiments. However, it is less common than solutions incorporating acetic acid. No difference in Western blotting sensitivity was observed between the blots that were Ponceau S stained and blots that were not stained with Ponceau S. We also performed Western blots to determine if different acids affect the sensitivity of Western blots (Figure 9C). JMV?M.Q8HTgNCT`:9 H.3&HOVdVEX#R-]py ^1=! These lower Ponceau S concentrations produced images of statistically similar sensitivity to that of the 0.1% (w/v) Ponceau S. Since most commercial preparations utilize 0.1% (w/v) Ponceau S; these results suggest that commercial stains are at least 100 times more concentrated than necessary for satisfactory results. 8600 Rockville Pike Empirical Formula (Hill Notation) C22H12N4Na4O13S4. Effect of acetic acid concentrations on protein staining intensity. 0000002746 00000 n trailer <]/Prev 342729>> startxref 0 %%EOF 78 0 obj <>stream % The use of 0.01% (w/v) Ponceau S in 1% (v/v) acetic acid instead of the commonly used Ponceau S stains will also reduce the cost of total protein staining. Our research suggests a standard concentration of 0.01% (w/v) Ponceau S in 1% (v/v) acetic acid used for 2 mins staining could be adopted by the scientific community to increase the rigor and reproducibility of Western blotting normalization. We investigated the effect of 0.1% (w/v) Ponceau S and 1% (v/v) acetic acid incubation time on the detection of 4g and 10g of cytosolic rat liver lysate (Figure 1). Effect of Ponceau S concentrations on protein staining intensity. The specified Ponceau S staining solution band intensities were calculated relative to the band intensities generated from a commercial Ponceau S stain (Amresco). The blocked strips were then incubated with primary antibodies PSMA6 (1:20,000, Abcam, catalog # ab109377, lot # GR60925-6) or PSMA3 (1:3000, clone MCP257, Biomol International, catalog #PW8115, Batch #z05821b) in 1% non-fat milk overnight at 4C. Significant differences between samples were calculated using one-way analysis of variance (Anova) (P < 0.05, Sigma Plot 11.0).