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    Protein Extraction Buffer

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    Rabia Anjum
    This document provides recipes for two protein extraction buffers. The first buffer contains Tris-Cl, NaCl, EDTA, glycerol, PMSF, DTT, and NH4Cl. The second buffer uses a phenol-based extraction followed by precipitation with ammonium acetate in methanol and washing with acetone. It involves resuspending samples in Tris-buffered phenol and SDS buffer, extracting the phenol layer after centrifugation, and precipitating proteins with ammonium acetate in methanol followed by washing and drying.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd

    Protein Extraction Buffer

    Uploaded by

    Rabia Anjum
    51 views1 page
    This document provides recipes for two protein extraction buffers. The first buffer contains Tris-Cl, NaCl, EDTA, glycerol, PMSF, DTT, and NH4Cl. The second buffer uses a phenol-based extraction followed by precipitation with ammonium acetate in methanol and washing with acetone. It involves resuspending samples in Tris-buffered phenol and SDS buffer, extracting the phenol layer after centrifugation, and precipitating proteins with ammonium acetate in methanol followed by washing and drying.

    Original Description:

    Protocols for protein extraction from roots

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    © © All Rights Reserved

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    This document provides recipes for two protein extraction buffers. The first buffer contains Tris-Cl, NaCl, EDTA, glycerol, PMSF, DTT, and NH4Cl. The second buffer uses a phenol-based extraction followed by precipitation with ammonium acetate in methanol and washing with acetone. It involves resuspending samples in Tris-buffered phenol and SDS buffer, extracting the phenol layer after centrifugation, and precipitating proteins with ammonium acetate in methanol followed by washing and drying.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    51 views1 page

    Protein Extraction Buffer

    Uploaded by

    Rabia Anjum
    This document provides recipes for two protein extraction buffers. The first buffer contains Tris-Cl, NaCl, EDTA, glycerol, PMSF, DTT, and NH4Cl. The second buffer uses a phenol-based extraction followed by precipitation with ammonium acetate in methanol and washing with acetone. It involves resuspending samples in Tris-buffered phenol and SDS buffer, extracting the phenol layer after centrifugation, and precipitating proteins with ammonium acetate in methanol followed by washing and drying.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 1

    Protein Extraction Buffer (Bakhsh et al.

    )
    Component

    80ml

    40ml

    5ml

    8.4ml

    4.2ml

    0.525ml

    8ml

    4ml

    0.5ml

    5 M NaCl

    4.8ml

    2.4ml

    0.3ml

    2 M Tris-Cl
    0.7%
    Phenylmethylsulphonylfluoride
    (PMSF) dissolved in
    isopropanol
    NH4Cl

    0.4ml

    0.2ml

    0.025ml

    8ml

    4ml

    0.5ml

    428mg 214mg 26.75mg

    Dithiothreitol (DTT)

    140mg

    0.5 M EDTA (pH 8.0)


    Glycerol

    70mg

    8.75mg

    Protein Extraction Buffer (Rodrigues et al.)


    1. Tris-buffered Phenol
    2. SDS Buffer
    Components with Working
    Concentration
    0.1 M Tris (pH 8.0)
    2% SDS

    Stock
    Concentrations

    0.8ml

    8ml

    50ml

    1.5 M

    0.0533

    0.533

    3.33125

    10%

    0.16

    1.6

    10

    0.04

    0.4

    2.5

    40 mM

    0.0199

    0.199

    1.24375

    48%

    0.5

    31.25

    5% Mercaptoethanol
    1 mM PMSF (in isopropanol)
    30% Sucrose

    3. Ammonium Acetate
    0.7708g in absolute methanol (100ml)
    4. Acetone (80%)
    1. Pulverized samples (250 mg) were resuspended in 0.8 mL of Tris-buffered phenol, pH 8.0,
    and 0.8 mL of SDS buffer.
    2. The samples were vigorously vortexed for 10 min and centrifuged at 16,000 g for 5 min at 4
    C
    3. The top phenol layer (0.5 mL) was transferred to a new tube.
    4. Proteins were precipitated for 2 h at 20 C with three volumes of pre-cooled 0.1 M
    ammonium acetate in absolute methanol.
    5. Pelleted by centrifugation (16,000 g for 5 min at 4 C)
    6. The pellet was washed once with pre-cooled 0.1 M ammonium acetate in methanol and
    once with pre-cooled 80% v/v acetone, followed by vacuum drying.

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