Improved Immunoblot by Brief Application of Low Dose Paraformaldehyde

Transcription

1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ * protein immunoblot Western blot 0 4% 0 4% 30 min Q-33 Improved Immunoblot by Brief Application of Low Dose Paraformaldehyde 1 Zhejiang University Hangzhou SUN Zhong-Quan 1 LU Wen 1 AI Heng 2 * China 2 Zhejiang Medical College Hangzhou China Abstract Protein immunoblot Western blot has been a widely adopted method for studying expression and posttranslational modification of proteins in cells and tissues Previous studies have shown that application of low concentration 0 4% of paraformaldehyde PFA an additional step specifically inserted between transferring and blocking the membrane can improve detection of target protiens in immunoblot In our research to find out the most effective concentration and time course of PFA treatment in immunoblot a series of concentration of PFA and time course of treatment were set up in experiments for detecting various proteins In general it is sufficient to improve detection in immunoblot by low concentration 0 4% and 30 minutes of application of PFA However fixation of proteins of large molecule weight did not manifest any improved effect for detection while fixation of medium and small proteins showed significant improvement Surprisingly fixation of exogeneous GFP which is a 28 kd protein expressed in HEK293 cells displayed no significant improvement In conclusion our study suggests that fixation of proteins of medium and small molecule weight by PF will improve the detection efficiency Key words protein immunoblot paraformaldehyde fixation No Y No 2013B01 * Tel aiheng@ zju edu cn Received June Accepted August Supported by Department of Education of Zhejiang Province No Y and Start-up Fund of Zhejiang Medical College No 2013B01 * Corresponding author Tel aiheng@ zju edu cn Co-first author

2 protein immunoblot Western blot 0 4% SDS-PAGE Table 1 nitrocellulose PVDF 1 h glutaraldehyde 6 TBST 2-4 paraformaldehyde Tween-20 PVDF % PBS 2011 Lee α- α-synuclein Table 1 < 20 Table 1 Proteins and corresponding antibodies kd Protein Molecular weight Species Antibody source GluN2B endogenous in brain kd Mouse monoclonal antibody Homemade GluA1 endogenous in brain 110 kd Rabbit monoclonal antibody Epitomics Shp-2 endogenous in brain kd Rabbit polyclonal antibody Santa Cruz GFP transfection into HEK293 cells 28 kd Rabbit polyclonal antibody Abcam BDNF endogenous in brain 14 kd Rabbit polyclonal antibody Santa Cruz α-synuclein endogenous in brain 17 kd Mouse monoclonal antibody Santa Cruz 1 2 HEK293 HEK293 ATCC 10% FBS Gibco 1% Gibco 1 mg /m DMEM Gibco 4 5g /L L RIPA 50 mmol /L Tris- 3 ~ 4 d 1 Lipofectamine TM ~ 300 g SD 10% HCl ph mmol /L NaCl 1% Triton-X Invitrogen 0 5% HEK g 60% ~ 70% pegfp-n1 5 min g Clotech min HEK293 OPTI-MEM Gibco PBS h 60 mm 1 ml RIPA 10% FBS 1% DMEM 36 ~ 48 h Pierce BCA

3 SDS-PAGE ~ 4 One way ANOVA Post hoc P μg 10 μg 12% ~ 15% SDS-PAGE 2 Mini-Protein 7 0 mm 8 3 mm 1 5 mm Bio- Rad 100 V 30 min 2 1 α- 120 V 0 4% α- 90 V 1 h α-synuclein 6 PBS 30 min 5% BSA TBST 1 h 0 1% ~ 0 8% 30 min 4 TBST 5 10 min HRP Pierce 1 h TBST 5 10 min ECL Pierce Quantity One Fig 1 Graphpad Prism 5 0 α- Fig 2 0 1% 0 4% α % 161 7% ± 22 6% Fig 2 Low dose of PFA treatment enhanced Fig 1 Schematic chart of the procedures of the detection of α-synuclein in immunoblot The experiment performed in this study The lysate of rat cortex of two concentrations 5 μg and 10 membranes were fixed by low dose of PFA immediately μg was subjected to SDS-PAGE with low dose of PFA after transferred Then the membranes were blocked by treatment for 30 minutes before normal Western blot 5% BSA We always incubated membranes with primary procedure PFA treatment of all concentration antibodies at 4 overnight and with secondary dramatically enhanced the detection of α-synuclein antibodies for 1 hour at room temperature The while internal loading control of β-actin without PFA membranes were visualized by ECL treatment remained comparable Up panel is the representative figure of immunoblot of α-synuclein 1 5 Down panel is the statistical results of the immunoblot Quantity One * P 0 05 Error bars indicate SEM n = 3

4 ~ 180 kd GluN2B 0 8% Fig 3A 94% ± 17 7% 0 1% 0 4% GluN2B 165 ~ 180 kd NMDA N-methyl-D-aspartic acid GluA1 GluN2B 110 kd AMPA 2-amino- 3-3-hydroxy-5-methyl-isoxazol-4-yl propanoic acid GluA1 92 7% ± 2 6% 0 4% 0 1% ~ 0 8% 2 Fig 3B 93 7% ± 5 3% 0 1% 92 3% ± 11 9% 0 8% Fig 3 Low dose of PFA treatment did not improve detection of GluN2B and GluA1 in immunoblot The lysate of rat cortex of two concentrations 5 μg and 10 μg was subjected to SDS-PAGE in addition with low dose of PFA treatment for 30 minutes before normal Western blot procedure A PFA treatment of all concentration did not show any detectable improvement of the detection of GluN2B whose molecular weights were 180 kd Up panel is the representative figure of immunoblot of GluN2B Down panel is the statistical results of the immunoblot * P 0 05 B The detection of GluA1 which was 110 kd did not improve by PFA Up panel is the representative figure of immunoblot of GluA1 Down panel is the quantification results of the immunoblot * P 0 05 Error bars indicate SEM n = % ± 62 3% 0 8% BDNF brain-derived neurotrophic factor BDNF 12 SHP- 2 Src homology domain 2 SH2 -containing BDNF Fig tyrosine phosphatase SHP-2 72 kd 4B 0 1% BDNF % ± 30 6% 0 4% SHP-2 BDNF 151 3% % ± 36 4% Fig 4A 0 8% BDNF 1 0 4% 134 3% ± 10 3% 210% ± 34 5% 0 1% % ± 34 3% 0 4%

5 Fig 4 Detection of proteins of medium and small molecular weight were significantly improved by PFA treatment The lysate of rat cortex of two concentrations 5 μg and 10 μg was subjected to SDS-PAGE including low dose of PFA treatment for 30 minutes before normal Western blot procedure A Immunoblot of SHP-2 which is a 72 kd protein was improved significantly Up panel is the representative figure of immunoblot of SHP-2 Down panel is the statistical results of the immunoblot ** P 0 01 B Low dose of PFA treatment also enhanced the detection of BDNF 14 kd in immunoblot Up panel is the representative figure of immunoblot of GluA1 Down panel is the quantification results of the immunoblot * P 0 05 Error bars indicate SEM n = 4 HEK293 3 GFP Fig 5 GFP 28 kd 70 ~ PVDF % 0 1% 4 20 ~ 60 min Fig 6 30 min BDNF 139% ± 12 3% 20 min 189% ± 12 1% 30 min 134% ± 11 4% 60 min 5 6

6 Fig 5 Detection of GFP expressed in transfected HEK293 cell was worsened The lysate of HEK293 cells of two concentrations 5 μg and 10 μg was subjected to SDS-PAGE in addition with low dose of PFA treatment for 30 minutes before normal Western blot procedure Treatment of PFA decreased the band intensity of GFP in membranes no matter application of any concentration of PFA The top of the figure is the representative immunoblot of GFP and the bottom of the figure is the quantification of three independent results * P 0 05 Error bars indicate SEM n = 3 Fig 6 Time course of fixed concentration PFA showed treatment of 30 minuets was sufficient to improve the detection The lysate of rat cortex of two concentration 5 μg and 10 μg was subjected to SDS-PAGE with low dose of PFA treatment before normal Western blot procedure We set the concentration of PFA to 0 1% and the time of treatment of PFA ranging from 0 minutes to 60 minutes the results herein suggested 30 minutes treatment was sufficient to improve the detection of BDNF The top of the figure is the representative immunoblot of BDNF the bottom of the figure is the quantification of three independent results * P 0 05 Error bars indicate SEM n = 3 Lee 6 Tween ~ 180 kd GluN2B 110 kd GluA1 0 1% 0 4% References 30 min 1 Renart J Reiser J Stark G R Transfer of proteins from gels to GFP GFP GFP metallothioneins-1 5% BSA TBST 1 h diazobenzyloxymethyl-paper and detection with antisera a method for studying antibody specificity and antigen structure J Proc Natl Acad Sci U S A Van Eldik L J Wolchok S R Conditions for reproducible detection of calmodulin and S100 beta in immunoblots J Biochem Biophys Res Commun Mizzen C A Cartel N J Yu W H et al Sensitive detection of -2 and -3 in tissue homogenates by immunoblotting a method for enhanced membrane transfer and retention J J Biochem Biophys Methods

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