The membrane was incubated for 1 hour at room temperature with goat anti-rabbit IgG peroxidase (HRP) (1:10,000, cat. Our results suggest that the addition of sulfosalicylic acid did not enhance the protein detection. Significant differences between samples were calculated using one-way analysis of variance (Anova) (P < 0.05, Sigma Plot 11.0). Ponceau S stain did not affect Western blotting sensitivity. If a lower background is needed wash with 1-5% acetic acid for 5-10 minutes. 0o0`$Kx(Lz-h}LEJ6h.G_HONh?$i(`azxdDXB>$'L##hA#(8`%Qje will also be available for a limited time. Hence, the use of total protein staining for Western blotting normalization is likely to increase significantly over the next decade. 4g and 10g protein samples were tested through multiple replications and revealed that there was no significant difference in protein band intensity at differing acid types and concentrations. hb```f``g`c` B,@Q&?%%yL86aXg40,G2k4egc Z0Q9GDBa/PUV\40,l`Zpm 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. 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. FOIA 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). 0000037392 00000 n
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jFg;9j/Nxwn)J0-dX.bk3%E`9~(S Using Sigma Plot 11 (Systat Software, Inc. CA), the results were graphed with raw and normalized data. Before 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. sharing sensitive information, make sure youre on a federal Effect of different Ponceau S concentrations in 3% TCA on protein staining intensity. 0000001860 00000 n
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. After incubation, the membrane strips were washed three times in TBST for five minutes. 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). Though Ponceau S is considered non-toxic to humans in small concentrations, the effect of being exposed to larger amounts of Ponceau S over time is unknown. 0000012169 00000 n
Effect of sulfosalicylic acid on Ponceau S protein staining intensity. To determine which current Ponceau S stain had the highest sensitivity for detecting proteins, we searched research papers, online recommendations, and company websites for Ponceau S stain recipes. 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. All further experiments were carried out using two minutes of staining time to allow the experimenter enough time to adequately stain and image several images at the same time. 0000013436 00000 n
Empirical Formula (Hill Notation) C22H12N4Na4O13S4. To determine if concentrations less than 0.1% (w/v) of Ponceau S would also be suitable for staining, experiments using 0.01 and 0.001% (w/v) Ponceau S were carried out (Figure 3). Effect of TCA and sulfosalicylic acid on protein staining intensity. And third, stripping and reprobing is often used to first detect the target protein and then detect the housekeeping protein. 0000006499 00000 n
4g and 10g of liver samples were loaded onto Bio-Rad Midi Criterion TGX Precast sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE, 4-20%, catalog #5671095). The methods recommended to stain and destain membranes with Ponceau S stain is also variable (Table 1). 0.1% Ponceau S (w/v) and 5% (v/v) acetic acid. Each experiment shown was carried out three independent times and repeated by at least two investigators. tNdpM|}b.u"wSNm
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We offer you a combination of products, technical know-how and excellent service. Membranes were incubated with Clarity substrate in the dark for 2 minutes. 0000013499 00000 n
The lower percentage of Ponceau S staining solutions are better for the environment and for general safety. Band intensities were totaled for each lane. Ponceau S solid was purchased from Alfa Aesar (MA, catalog# J60744), Trichloroacetic acid (TCA, catalog# UN1839, Fisher Scientific, PA), Sulfosalicylic Acid (SA, catalog# {"type":"entrez-nucleotide","attrs":{"text":"LC255201","term_id":"1345669231","term_text":"LC255201"}}LC255201, LabChem, PA), Acetic Acid (catalog # UN2789, Fisher Scientific, PA), Bovine Serum Albumin (BSA, catalog# A7030, Sigma-Aldrich, MO). After 2 minutes of staining, the membranes were briefly washed with ddH2O water for 20 seconds. 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. 0000037639 00000 n
Electrophoresis was carried out at 120V for 70 minutes or until dye front reached the bottom of the gel. )K]aVn#^15K7{U
/.Mf`DEU&bZ. As such, understanding which Ponceau S stain is optimal for total protein detection is needed. :v= 0;GOT &*8s3u@ Y+LoXCJkf*kJ!F-In:1y(B(
+K\ }MfR82o/5 L&X` X*Mptd2)@clRE~_?`zv&Q*-GE+{2su6o#O\E"d39J@]g"uU'J0?.]C6 #4CdJ{&|?~..yQehdOn&"2Y&y&*e'e2+xtF2AC[&0Tumjh8viR!Ts88v{9>21G`*Y81 $3mJzOwwX?F-qdzfeV-G|$A!oy=zF Q
-UjvIv ! Hence, our findings suggest that the large variations in Ponceau S stains with different concentrations of Ponceau S and different concentrations of acids as well as different combinations of acids are unnecessary. Then, rinse the membrane with running distilled water for 2-3 minutes [. To further complicate the use of Ponceau S, scientists have also used other compositions of Ponceau S stain including 0.6% (w/v) Ponceau S in 3% (w/v) TCA [19]. 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]. 0000037986 00000 n
The acetic acid concentration did not significantly affect the sensitivity of the Ponceau S staining. A standard Ponceau S staining solution containing less Ponceau S than is commonly employed should be utilized by the scientific community. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. Imaging of the membrane was conducted by the Chemidoc MP (Bio-Rad). 0000008219 00000 n
I Quantification of 4g and l0g of proteins stained with 1% (v/v) Acetic Acid and 0.1% (w/v) Ponceau S for 1, 2, 5, or 10 minutes. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. The supernatant was then removed, transferred in new tubes and quantified. 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 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. Westburg is an independent distributor providing high quality, innovative products to Life Sciences laboratories. For housekeeping protein normalization, housekeeping proteins such as -actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or -tubulin, which are expressed constitutively at relatively high levels, are probed together with the target protein using different antibodies [3; 4; 5]. 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. Westburg offers this product in The averages and standard deviations were found for each set of experiments. The staining is reversible and compatible with subsequent Western blotting [, Incubate membrane in Ponceau S solution for 5-10 minutes at room temperature (RT). 0000006891 00000 n
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# A9044, Sigma-Aldrich,) secondary antibody in TBST containing 1% non-fat milk. To determine if the sensitivity of protein detection was affected by changing the concentration of Ponceau S in 3% TCA, 0.1%, 0.2%, and 0.5% (w/v) Ponceau S in 3% TCA was investigated (Figure 6). 0000012074 00000 n
PMC legacy view aDepartment of Neurobiology, Physiology, and Behavior, University of California, Davis, CA, US, bDepartment of Physiology and Membrane Biology, University of California, Davis, CA, US. To effectively determine the relative amount of a target protein on a blot, a normalization control is needed. This work was partly funded by AHA 16GRNT31350040 and NIH 5P42ES004699. Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Non-validated antibodies often result in artifactual results [3]. Learn more 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. The stained blots obtained using the different Ponceau S compositions were normalized to the blots obtained using the commercial Ponceau S solution. 0000001973 00000 n
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official website and that any information you provide is encrypted Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant. 0000002746 00000 n
Most protocols suggest that 5 minutes of Ponceau S staining is optimal. Luxembourg, Last chance: Lonza classical cell culture media special prices, Fast-Blot Semi-dry Western Blotting System, Biometra Eco Tankblot Western blot transfer systems, Pink/red protein bands will start to appear. 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. Effect of TCA on Ponceau S protein staining intensity. To normalize the Ponceau S staining results we utilized a commercial Ponceau S solution from Amresco. 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. Staining of these membranes with three different Ponceau S stains showed that the detection sensitivity of Ponceau S for BSA was the same for all stains (Figure 9A). 0000006018 00000 n
Several total protein stains are available including Ponceau S and Stains Free Stains [8; 9; 10]. While total protein normalization offers many advantages over housekeeping protein normalization [1; 2; 5; 8], analysis of Western blotting papers published in January 2018 on PubMed showed that > 96% of manuscripts, that included data for normalization of Western blots, use a housekeeping protein for normalization of Western blots (using manual tabulations of all manuscripts containing Western blotting). Gilda JE, Ghosh R, Cheah JX, West TM, Bodine SC, and Gomes AV, Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS). We were unable to find an optimal Ponceau S stain as all the stains worked equivalently. To independently validate that 0.1% (w/v) Ponceau S with 1% (v/v) acetic acid was as good as any of the other stains, SDS-PAGE was carried out using 75ng to 2g of purified bovine serum albumin (BSA) and subsequent protein transfer to nitrocellulose membrane. 5% acetic acid and 30% SA did not affect the Western blotting sensitivity when compared to unstained membranes. 0000012312 00000 n
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The desired acid was measured and added to the Ponceau S solution and vortexed. 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]. Using the Trans-blot Turbo Transfer System (Bio-Rad), the proteins on the gel was transferred to nitrocellulose membrane (7 minutes). YA,.&`H#Qg4f6yAOPg+&htf&>WJCa R +09$bAiAf:[b]]qCtSH! 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. To prepare the strips for Western blotting, the strips were once again destained with TBST. The staining intensity of solutions containing TCA was similar to when acetic acid was used (Figure 5). 0000011485 00000 n
How many human genes can be defined as housekeeping with current expression data? The strips were then imaged using the Chemidoc MP. 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. Quantification of 4g and 10g of protein stained with 0.1% (w/v) Ponceau S with 1% (v/v) acetic acid, 2% (w/v) Ponceau S with 30% (v/v) sulfosalicylic acid, and 2% (w/v) Ponceau S with 30% (v/v) sulfosalicylic acid and 30% (v/v) trichloroacetic acid (TCA) for 2 minutes. The Fisher Scientific list price of 10g of Ponceau S is $55.50, and the list price of 500ml of glacial acetic acid is $52.66 for 500ml. Heart and circulatory physiology, Western blotting using in-gel protein labeling as a normalization control: stain-free technology, (CAS 6226-79-5 (solid)) Santa Cruz Biotechnology, Staining membrane-bound proteins with ponceau s, Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Quantification of 4g and 10g of protein stained with 1% (v/v) acetic acid and either 0.1%, 0.2%, or 0.5% (w/v) Ponceau S for 2 minutes. 0000004011 00000 n
It is easily reversed with repetitive washings in water [, Immerse membrane in Ponceau S staining solution for 5 minutes. ?Q'v?vr29ozcCEev0q; !zG`@%"(b\Qp=zzDkznGz|f #p='Fz p=GXcczf=z\c=z\g~B\Rz~BA0_ 6i zd=zb.QA&K;3_oWntsT: q${3L~lh;\.H[6Nn0Rxu]oNWW4mtBBWBOQO=98qC 4R>Z_N~`Fa|U?]Mo_?_xZ^/^ U2GpYHi~#]khzfyWMz$7pFNFtl53+1TLB=+d&C$Ob~hu[/rV/T3zD~f;[W[jRQL>y7 #QyO.^m^5f@*JZ' :yVt_AOcP\hGa&vK0v4K_NTr7zAI"&+$__|DR7_8|CS7j[
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,/#-:=t$]f#ikgwgI5K =g_k%i>?#3h 1R,^0!?k=l57A6FYoB6jIj[fl;f>XkwdyR+! Salinovich and Montelaro were the first to report the use of Ponceau S for membrane staining [13]. Using less of any compound, even non-toxic, is good for the environment. Membranes were stained with the different 3mL of Ponceau S solutions for 2 - 10 minutes on the shaker (100 rpms) at room temperature and then rinsed with distilled water to remove background stain. Federal government websites often end in .gov or .mil. These errors, which can lead to incorrect interpretation of the data, are reduced by using a normalization control. % Effect of Ponceau S incubation time on protein staining intensity. Destain in water until the background becomes clear [, Ponceau S in a specially formulated acetate buffer, Ponceau S stain is designed for rapid (5 min) staining on nitrocellulose or PVDF membranes [, Rapid (5 min) staining of protein bands on PVDF or nitrocellulose membranes. Effect of different Ponceau S concentrations on protein staining intensity. Normalization of Western blotting data is a critical step that is needed to reduce errors caused by unequal sample loading across lanes in a gel, inconsistent sample preparation, and variations due to experimental errors. {_ r3Dmc\9lN&M]rsVq^VgZAJ(`(LvmL p*C(3
mcB!E0JIBi'Z@G,b}I lbEz For Western blot detection destaining is not required. Homogenization buffer was used as the blank. Wash the membrane 2-3 times with DI water for 5 minutes each [, 0.1% (w/v) Ponceau S and 5% (v/v) acetic acid, Wash the membrane briefly in TBST and stain the transfer membrane in staining solution for 5-10 min at RT. The images taken were quantified with Image lab 5.1 software. Belgium, Ponceau S is the most commonly used stain for total protein normalization. Transfer and staining of proteins in western blot, The effect of various stains on quantitative agarose-gel electrophoresis: Evaluation of the results with the aid of an analog computer, Ponceau S solution for electrophoresis (0.2 %), https://www.scbt.com/scbt/product/ponceau-s-solution-6226-79-5-solid, http://cshprotocols.cshlp.org/content/2007/4/pdb.rec10772.full?text_only=true, http://www.encorbio.com/protocols/blotting.htm/, https://www.abcam.com/protocols/transfer-and-staining-of-proteins-in-western-blot, https://advansta.com/products/protein-stain-AdvanStain-Ponceau/, https://biotium.com/product/ponceau-s-solution/, https://media.cellsignal.com/pdf/59803.pdf, https://www.gbiosciences.com/image/pdfs/protocol/786-575_protocol.pdf, https://www.novusbio.com/products/ponceau-s-staining-solution_nb5225, https://www.itwreagents.com/download_file/product_infos/A2935/en/A2935_en.pdf, https://rockland-inc.com/Report.aspx?id=50094, https://www.serva.de/enDE/ProductDetails/787_84_33427_Ponceau_S_Solution_for_Electrophoresis_0_2_percent_212_84.html, https://www.sigmaaldrich.com/catalog/product/sial/p7170?lang=en®ion=US&gclid=EAIaIQobChMI1PaRt6f74AIVix6tBh245Ao5EAAYASAAEgKquPD_BwE, Incubate the membrane in the ready-to-use AdvanStain Ponceau solution for 5 minutes or less to detect protein bands. trailer
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#A0545, Sigma-Aldrich) or anti-mouse (1:15,000 anti-mouse Cat. government site. 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. Concentrations of Ponceau S sold by Manufacturers. OwR%?6Yi1,L~v7-c0k;b7m7_dU
i@lH2TOD;UJdDgdqHF^q>N?0+QyG=|GKOq|_v? Briefly, PVP (0.1-1% (w/v)) or 1-5% (v/v) glycerol was added to 0.1% Ponceau S in 1% acetic acid and the protein staining intensity was found to be similar to the staining intensity after exposure to 0.1% Ponceau S in 1% acetic acid. We then systematically investigated the different Ponceau S stains. 0000014596 00000 n
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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]. Effect of Ponceau S concentrations on protein staining intensity. 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). 2 0 obj The new PMC design is here! 0000012894 00000 n
To determine if sulfosalicylic acid in the absence of TCA could enhance sensitivity, 2% (w/v) Ponceau S in 30% (v/v) sulfosalicylic acid was compared against 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid and no difference in sensitivity was observed (Figure 8). 0000014659 00000 n
HTTnT14H8U-=3^r|;ZQZYM_V>8^? After staining the blots with our different Ponceau S solutions we then destained the blots and restained all blots with the same commercial Ponceau S solution. 0000011370 00000 n
Proteomics grade Ponceau S in liquid form was purchased from Amresco (catalog# 97063, VWR, PA). As such, we carried out the Ponceau S staining experiments with total protein concentrations of 4g and 10g, which are at the low end of most total protein staining experiments and would require stains that can detect relatively low amounts of total protein. The membrane can be destained in minutes by incubating in water [. 0000014083 00000 n
The actual composition of the commercial Ponceau S is not provided by the manufacturer. 0000011990 00000 n
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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. Barber RD, Harmer DW, Coleman RA, and Clark BJ, GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Cold Springs Harbor recommends 0.5% (w/v) Ponceau S in 1% (v/v) acetic acid [16], EncorBio advises 0.25% (w/v) Ponceau S in 40% methanol and 15% acetic acid [17], while Abcam suggests 0.02% (w/v) Ponceau S in 0.3% (w/v) TCA and 0.3% (w/v) sulfosalicyclic acid [18]. 0000011925 00000 n
The rinsed strips were subsequently imaged using the Bio-Rad Chemidoc MP. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. A solution 0.02% (w/v) Ponceau S in 0.3% (w/v) TCA and 0.3% (w/v) sulfosalicylic acid was also previously utilized [18]. The lower blots show the same blots destained and then restained with commercial Ponceau S and the lower bar charts show the normalized results. There are three major concerns with the use of housekeeping proteins as normalization controls. 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). ![Sn `W# \;G]?H^ $}UWdlwi(
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As a service to our customers we are providing this early version of the manuscript. The samples were quantified using the nanodrop 2000C (Thermo Scientific) using BSA as a standard. Currently, a wide variety of Ponceau S formulations have been used to assess the quality of a transfer, locate proteins, and carry out normalization. {*Qg"~?>'YmIIP;XmZxJ>)
Rz1 To determine if TCA concentration had any effect on Ponceau S staining intensity in relation to the more commonly used acetic acid, 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid, 5% (v/v) acetic acid, and 3% (v/v), TCA were quantified against each other. Images were quantified using Image Lab software (Version 5.2.1, Bio-Rad). HHS Vulnerability Disclosure, Help Our results suggest that a simple and less expensive Ponceau S stain (0.01% (w/v) Ponceau S in 1% acetic acid (v/v)) gives the same sensitivity of protein detection as all the other formulations, suggesting that one Ponceau S stain could be utilized for Western blotting normalization. The following solutions were compared: 0.0001, 0.001, 0.01, 0.02, 0.05, 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid; 0.1% Ponceau S in 1%, 10%, or 20% (v/v) acetic acid; 2% (w/v) Ponceau S in 30% (w/v) sulfosalicylic acid, and 2% (v/v) Ponceau S in 30% (w/v) sulfosalicylic acid and 30% (w/v) TCA. After proteins have been visualized, rinse the membrane with DI and rapidly immerse in an aqueous solution of 0.1 M NaOH. For protein destaining, wash the membrane with 0.1N NaOH solution for 5 minutes and repeat once. pCHV_\~m+-=TBX&VSDF3?3r=X~6j3&oCzpU3o=>wW Effect of acetic acid concentrations on protein staining intensity. Errors shown are standard deviation of the mean (SEM). 0000000016 00000 n
A comparison between 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid and 2% (w/v) Ponceau S, 30% (v/v) TCA, and 30% (w/v) sulfosalicylic acid (Figure 8) showed no statistically significant difference for both 4g and 10g of sample. Quantification of 4g and 10g of protein stained with 1% (v/v) acetic acid and 0.1%, 0.01%, and 0.001% (w/v) Ponceau S for 2 minutes. The Amresco Ponceau S composition is not given by the manufacturer. C(=$ S'uy_B(0Q,VJ!r@!G!XDQpKBIQibgPT0$%il 7X38zH Each result was independently reproduced by at least two different investigators. Would you like to allow them? Western blots are usually carried out with total protein loading amounts in the range of 10-20g. Second, the housekeeping gene and protein levels can change during developmental phases and under different experimental conditions. The authors disclose no conflict of interest. Such binding is achieved by the negatively charged component of the dye attaching to the positively charged amino acids of proteins. 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. Ponceau S is likely to be one of the most commonly used stains for normalizing Western Blots in the near future. Quantification of 4g and 10g of protein stained with 0.1% (w/v) Ponceau S with 1% (v/v) acetic acid (AA), 0.2% (w/v) Ponceau S with 3% (v/v) TCA and 3% (v/v) sulfosalicylic acid (10% stock) and 0.02% (w/v) Ponceau S with 0.3% (v/v) TCA and 0.3% (w/v) sulfosalicylic acid (1% stock) for 2 minutes. Our initial experiments were carried out to determine if we can increase the sensitivity of the Ponceau S stain. Al-Amoudi MS, Salman M, Al-Majthoub MM x]Ms7We"vs%yW3G\(;dS"EyB5d:llY /_f~}}KW>qd]=#N.T6tKz0? Ponceau S solutions with 3% (v/v) TCA have been utilized in the literature [11]. 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. Lanes were manually fit to each lane and all bands including faint bands were detected. 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. The specified Ponceau S staining solution band intensities were calculated relative to the band intensities generated from a commercial Ponceau S stain (Amresco). %PDF-1.3 }Zt2-l5=EbkOG UEEuAr-wK,3D Rat livers were obtained from Pel-Freez Biologicals (Arkansas), Nitrocellulose (Trans-Blot Turbo Midi Nitrocellulose Transfer Packs #1704159, Bio-Rad). The Netherlands, et al. 0000005321 00000 n
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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.